Jbx482586.indd

482586JBX18710.1177/1087057113482586Journal of Biomolecular ScreeningShadrick et al.
Review Article Journal of Biomolecular Screening18(7) 761 –781 Discovering New Medicines Targeting
2013 Society for LaboratoryAutomation and ScreeningDOI: 10.1177/1087057113482586 Helicases: Challenges and Recent Progress
William R. Shadrick1, Jean Ndjomou1, Rajesh Kolli1,
Sourav Mukherjee1, Alicia M. Hanson1 and David N. Frick1
Abstract
Helicases are ubiquitous motor proteins that separate and/or rearrange nucleic acid duplexes in reactions fueled by
adenosine triphosphate (ATP) hydrolysis. Helicases encoded by bacteria, viruses, and human cells are widely studied
targets for new antiviral, antibiotic, and anticancer drugs. This review summarizes the biochemistry of frequently targeted
helicases. These proteins include viral enzymes from herpes simplex virus, papillomaviruses, polyomaviruses, coronaviruses,
the hepatitis C virus, and various flaviviruses. Bacterial targets examined include DnaB-like and RecBCD-like helicases. The
human DEAD-box protein DDX3 is the cellular antiviral target discussed, and cellular anticancer drug targets discussed
are the human RecQ-like helicases and eIF4A. We also review assays used for helicase inhibitor discovery and the most
promising and common helicase inhibitor chemotypes, such as nucleotide analogues, polyphenyls, metal ion chelators,
flavones, polycyclic aromatic polymers, coumarins, and various DNA binding pharmacophores. Also discussed are common
complications encountered while searching for potent helicase inhibitors and possible solutions for these problems.
Keywords
motor protein, ATPase, RNA binding proteins, molecular probes, antivirals, antibiotic, anticancer
Helicases are tiny molecular motors fueled by adenosine A PubChem Assay Identification (AID) number will also be triphosphate (ATP) hydrolysis that grab one strand of DNA noted for assays used to identify or characterize discussed or RNA and peel it from its complementary strand. In cells, helicase inhibitors.3 DNA helicases play key roles in DNA replication, recombi- There are many reasons why helicase inhibitor develop- nation, and repair. Cells need RNA helicases for transcrip- ment is challenging. We have encountered two basic problems tion, translation, and RNA splicing. More than 10 years ago, in our efforts to discover hepatitis C virus (HCV) helicase several potent antiviral drugs were discovered that inhibit inhibitors. First, high-throughput screens using assays moni- an essential herpes simplex virus (HSV) helicase complex, toring helicase-catalyzed nucleic acid duplex separation yield and this discovery inspired many others to study helicases few hits, and second, most of the hits act by binding the as drug targets.1,2 Discovering similarly potent and specific nucleic acid substrate. For example, the Scripps Research inhibitors for other helicases has been quite challenging, but Institute Molecular Screening Center tested 290,735 com- considerable progress has been made in recent years. This pounds in the National Institutes of Health (NIH) small- review discusses recent progress toward making helicases molecule collection using an assay that monitors the ability of more tractable drug targets. We discuss below data recently the HCV helicase to separate duplex DNA (PubChem published or deposited in the PubChem BioAssay,3 com- BioAssay AID 1800).11 Only 500 compounds (0.2%) were mon chemical scaffolds identified as hits in high-throughput confirmed as hits (AID 1943), the most potent hits were assay screens, how hits have been optimized, and novel new high-throughput assays. Because numerous other reviews on 1Department of Chemistry & Biochemistry, University of Wisconsin– helicase biochemistry, helicase assays suitable for screen- Milwaukee, Milwaukee, WI, USA ing, and the role of helicases in biology are available,4–9 we will only briefly review key points before discussing inhibi- Received Dec 1, 2012, and in revised form Jan 28, 2013. Accepted for publication Feb 22, 2013.
tor development in more detail. Throughout this article, helicase inhibitors will be identified by a PubChem Compound Identification (CID) number, which can be used David N. Frick, PhD, Department of Chemistry & Biochemistry, University of Wisconsin–Milwaukee, 3210 N Cramer St, Milwaukee, WI to access a wealth of other data for each compound by searching the CID in the PubChem Compound database.10 Email: [email protected] Journal of Biomolecular Screening 18(7) artifacts (AID 485301), and the most potent hits did not inhibit strand,32 whereas non–ring helicases do not form rings but HCV RNA replication in a cell-based assay (AID 463235). rather function as monomers,33 dimers,34 or higher order oligo- Many assay artifacts, or false leads, seen in helicase assays mers.35,36 Ring helicases consist of identical subunits, each of that monitor DNA duplex separation, such as the one used for which contains a domain that resembles the Escherichia coli HCV helicase, result from a compound's ability to interact protein RecA.37 ATP binds at the interface of two RecA-like with the helicase's DNA substrate. Such DNA binding com- domains such that there are six ATP binding sites on a hexa- pounds are still very difficult to identify in a high-throughput meric ring helicase. Sequential or concerted ATP hydrolysis format. Solutions to various helicase inhibitor development causes a ring helicase to spin down a nucleic acid strand.30 problems include extensive counterscreening, innovative Non–ring helicases38 consist of two RecA-like domains cova- assays monitoring helicases in cells or helicase interactions lently linked in tandem on the same polypeptide,39 and ATP with other proteins, and structure-based design. As discussed binds between these "motor domains."40 ATP binding and below, all these efforts have led to potent, specific, and some hydrolysis cause a non–ring helicase to expand and contract so drug-like helicase inhibitors.
that the helicase moves along DNA (or RNA) like an inch- The goal of this article is to discuss how new helicase worm.41–43 The above characterization likely oversimplifies inhibitors were discovered and optimized in the past few how helicases function as molecular motors, and exactly how years. These small molecules target proteins linked to these molecular machines assemble is still a subject of consid- diverse diseases, such as viral infections, bacterial infec- erable research and debate.
tions, premature aging, and cancer. Due to space limita- Both ring and non–ring helicases must first load on single- tions, this is not a comprehensive review of these subjects. stranded DNA (or RNA) before they can separate a duplex. Rather, we intend to update other helicase articles, such as Once loaded on single-stranded DNA (or RNA), most heli- Xu Guang Xi's review about helicases as antiviral and anti- cases move in either one of two possible directions. Some cancer drug targets.12 We have chosen to focus on how new move from the 5′-end to the 3′-end of the strand to which they helicase inhibitors were identified and optimized, common are bound, and others move in a 3′ to 5′ direction.44,45 screening problems, and the chemistry of common helicase In addition to movement directionality and oligomeric inhibitor chemotypes. Numerous other reviews are avail- state, helicases are also classified based on their genetic able that are focused on either viral helicases7,13 or cancer- similarities. All helicase genes evolved from the same com- linked helicases.14–16 Many excellent resources also cover mon ancestor, and helicase proteins share common signa- helicase biochemistry in more detail, but most are focused ture sequences indicative of family relationships. Helicase on specific helicases such as the ones encoded by HSV,17–19 families are then grouped into superfamilies.46,47 Most HCV,20–24 flaviviruses,25 or humans.16,26–28 members of helicase superfamily 1 (SF1)48 and superfamily 2 (SF2)49 are non–ring helicases, and members of super- Introduction to Helicase Structure and
family 3 (SF3) and superfamily 4 (SF4) are typically ring helicases.9 HSV and human coronaviruses (CoV)50 encode the SF1 helicases that will be discussed below. SF2 helicase Cells use helicases whenever they need to access DNA or drug targets to be discussed are the NS3 proteins encoded RNA, and all life forms encode helicases.4,5 The only excep- by HCV and related viruses, the cellular DEAD-box pro- tions are some viruses that replicate in a cell's nucleus, where teins,51 and human RecQ-like helicases.52 SF3 helicases dis- they might hijack cellular helicases to access or copy genetic cussed below include viral DNA helicases encoded by material.8 DNA helicases separate the two strands of the dou- human papillomaviruses (HPVs)53,54 and polyomaviruses ble helix when it is copied, repaired, or transcribed into RNA. (e.g., simian virus 40 [SV40]).31 All SF4 helicases discussed Cells need RNA helicases for messenger RNA (mRNA) tran- below, as targets for new antibiotics, resemble the E. coli scription, translation, and to assemble or disassemble RNA- DnaB hexamer, which unwinds DNA and coordinates lead- protein complexes such as the ribosome. Viruses with RNA ing and lagging strand DNA replication.55 Many other heli- genomes also use helicases to resolve RNA duplexes formed cases in other helicase superfamilies (i.e., Rho-like helicases after replication. In other words, helicases guard access to our in superfamily 5 and the MCM proteins in superfamily 6)9 genomes. As genome guardians, helicases are linked to a myr- and the related AAA+ superfamily47 could someday be iad of disorders caused by abnormal gene expression, cell pro- important drug targets, but they will not be further discussed liferation, and infectious pathogen replication.19,27,29 here because specific small molecules that inhibit them RNA and DNA helicases are complex molecular motors have not yet been reported in the literature.
that use ATP to fuel nucleic acid base pair separation and/or rearrangement.6,9 On the molecular level, most helicases grab Helicases as Drug Targets one strand of DNA or RNA and move along it to displace its complement. Some helicases assemble into oligomeric rings The primary motivation to discover potent and specific (typically hexamers),30,31 which encircle one DNA (or RNA) helicase inhibitors is to control the ability of an organism to Shadrick et al. access genetic material. In theory, one could use helicase Virus-Encoded DNA Helicases inhibitors to control any aspect of gene replication or expression, but the goal of most present efforts is to find As noted above, only helicase inhibitor–based drugs target helicase inhibitors that simply prevent the replication of an HSV helicase. HSV is in the family Herpesviridae, mem- infectious pathogens or cancer cells. Antibiotics could be bers of which cause chickenpox/shingles (Varicella zoster), developed from potent and specific inhibitors of bacterial cold sores (HSV-1), and genital herpes (HSV-2). HSV-2 helicases, such as the DnaB55 protein that acts at bacterial infects 40 to 60 million people worldwide, with 1 to 2 mil- replication forks, or proteins involved in recombination, lion cases each year, and typically spreads through infected such as RecBCD.36 Inhibitors of cellular helicases could mucosa.65 All HSV are fully enveloped double-stranded function as antivirals or be used to control cancer cells or DNA viruses, which encode seven proteins needed for DNA make them more sensitive to chemotherapy.15 replication: the origin binding protein UL9, the single-stranded DNA binding protein ICP8, a heterodimeric poly-merase (UL30 and UL42), and a heterotrimeric helicase-primase complex (UL5, UL8, and UL52). UL52 is Much of what we know about helicases comes from studies the primase, and UL5 is the helicase. UL8 coordinates heli- performed with proteins first purified from benign E. coli labo- case-primase activity with the help of ICP8 and assists heli- ratory strains, such as the E. coli helicase that coordinates DNA case processivity. UL5 is an SF1 helicase, and it unwinds in replication, called DnaB.55,56 Inhibitors of E. coli helicases a 5′ to 3′ direction, but only in the presence of UL52.66,67 could be used, however, to treat pathogenic strains of E. coli, Papillomaviruses and polyomaviruses encode the other which cause more than 100 million gastrointestinal infections viral DNA helicases that are widely studied as drug targets. each year and about 170,000 deaths.57 Like E. coli, other gram- Both families have smaller DNA genomes than the viruses negative pathogenic bacteria, such as Pseudomonas aerugi- in Herpesviridae, and their circular genomes encode few nosa, encode an SF4 DnaB-like helicase that they use to proteins other than an SF3 helicase. Host DNA polymerases coordinate leading and lagging strand DNA replication. P. and other replication proteins assemble around the viral aeruginosa causes pneumonia, urinary tract infections, and SF3 helicase to synthesize new viral DNA in the same bidi- sepsis.58 Gram-positive bacteria encode DnaB-like proteins rectional semi-discontinuous manner used by most cellular that have been targeted to find treatments for Bacillus anthra- organisms. The prototype polyomavirus, SV40, was acci- cis, the causative agent of anthrax, and Staphylococcus aureus. dentally introduced to humans in the polio vaccine, but it S. aureus causes many natural and hospital-acquired infec- has not yet been clearly linked to any human disease. SV40 tions, which typically respond to current antibiotics.58 is closely related to two human polyamaviruses, which However, new S. aureus drugs are desperately needed because cause debilitating illnesses in immunocompromised of the evolution of methicillin-resistant S. aureus, which is patients. One is the BK virus, which is named after the resistant to penicillin and other beta-lactam antibiotics.59 transplant patient from whom it was first isolated, and the The other bacterial helicase targets discussed here are other is the John Cunningham virus (JCV).68 The polyoma- the non–ring helicases that form the multifunctional virus SF3 helicase is the large tumor antigen (TAg), a well- RecBCD complex.60 The RecBCD complex prepares DNA conserved viral protein transcribed soon after infection.69 for homologous recombination and/or repair. RecB and TAg contains an N-terminal J-domain, which stimulates the RecD are both SF1 helicases, which move in opposite direc- ATPase activity of the Hsp70 chaperone, a central origin– tions, on complementary strands, to drive the translocation binding domain, and a C-terminal helicase domain fueled of the RecBCD complex along DNA.61 A separate nuclease by ATP hydrolysis. Like other replicative ring helicases, function of RecB degrades both strands of DNA as the com- TAg forms two hexamers at the origin of DNA replication plex translocates. Upon reaching a Chi recognition that move in opposite directions as the replication bubble sequence, RecB stops cleaving the 3′-strand but continues opens and DNA is copied.31 to digest the 5′-strand, leaving an extended 3′ overhang of Members of the family Papillomaviridae have a circular single-stranded DNA (ssDNA).62 RecA binds the 3′ over- genome like SV40, but the papillomavirus genome is larger hang and helps it invade a homologous duplex to form a and more complex. More than 120 different strains of HPV DNA crossover to be resolved at a Holliday junction.37 infect more than 440 million people, and they cause about RecBCD also destroys bacteriophage DNA that lacks Chi 250,000 deaths per year. Most HPV strains cause genital sites, unless the virus encodes a protective protein.63 Most warts, but several pathogenic variants, such as HPV16, cause work with RecBCD has been done with the E. coli complex, cervical cancer.70 The HPV helicase is the viral E1 protein, but pathogenic bacteria, such as the ulcer causing which has both site-specific binding activity and unwinding Helicobacter pylori, encode RecBCD homologues. The H. activity. Like TAg, E1 is an SF3 ring helicase with many pylori RecBCD homolog, which will be discussed later, is functions and domains: a C-terminal helicase/ATPase domain, a central origin DNA binding domain, and an Journal of Biomolecular Screening 18(7) N-terminal regulatory domain. The N-terminal E1 region three times the size of HCV. Coronaviruses typically cause contains a nuclear localization signal, a nuclear export signal, upper respiratory infections (i.e., the common cold), but a conserved cyclin-binding motif, and several phosphoryla- some species, such as severe acute respiratory syndrome tion sites. E1 hexamers form at each replication fork at either coronavirus (SARS-CoV), can cause life-threatening pneu- end of a replication bubble. Exactly how the hexamers coor- monia. Another deadly coronavirus was recently isolated dinate their activity either by interacting with each other or from patients in Saudi Arabia.86 The SARS-CoV helicase is other proteins at the replication fork is still a subject of inves- nonstructural protein nsp13.50 Like NS3 from flaviviruses, tigation. The internal DNA binding hairpins of each subunit the SARS-CoV helicase has RNA 5′ triphosphatase form a spiral staircase, and these hairpins pull the DNA through the center of the ring one nucleotide at a time, such that six ATP molecules are used to move six nucleotides.53 Cellular Helicases as Antiviral Targets RNA Helicases Needed for Virus Replication Viruses that enter the nucleus often hijack cellular helicases during viral replication. For example, the human cellular Two classes of RNA helicases have been studied as drug helicases DDX1, DDX3, DDX24, MDA-5, RNA helicase A targets: helicases encoded by viruses and cellular helicases (RHA), and Werner syndrome protein (WRN) have been needed for virus replication. The most widely studied viral linked to human immunodeficiency virus (HIV) replication. helicase is the nonstructural protein 3 (NS3) encoded by All six are SF2 helicases. RHA was the first cellular heli- HCV. NS3 is an SF2, non–ring, 3′ to 5′ helicase. HCV is the case linked to HIV replication88 and is encapsulated in the only species in the genus Hepacivirus, which is part of the HIV particle.89 The melanoma differentiation-associated family Flaviviridae.71 The genus Flavivirus, as well as its gene 5 (MDA-5) helicase was called "RH116" in the study numerous important human pathogens such as yellow fever linking it to HIV replication.90 A dominant negative heli- virus (YFV), Japanese encephalitis (JEV), West Nile virus, case-defective WRN allele inhibits HIV long terminal and dengue virus, is also part of Flaviviridae.25 All flavivi- repeat transactivation and HIV replication.91 DDX3 facili- ruses encode NS3 proteins similar in form and function to tates the export of HIV RNA transcripts from the nucleus the HCV helicase.25,72–74 NS3 proteins are multifunctional, into the cytoplasm.92 DDX1 restricts HIV-1 Rev function in with the N-terminal domain functioning as a protease human astrocytes,93,94 and DDX24 helps with HIV packag- needed for viral polyprotein processing and the C-terminal ing.95 Although all the above helicases could serve as anti- domain functioning as a helicase. HCV and related viruses viral drug targets, small-molecule inhibitors have been encode the only proteins known that are both proteases and found so far only for DDX3, as will be discussed in more helicases. NS3 proteases are active only when combined detail below.
with another viral peptide. The HCV NS3 protease is active only when combined with HCV nonstructural protein 4A Cellular Helicases as Targets for Cancer (NS4A),75,76 and NS3 from flaviviruses is activated by non- structural protein 2B (NS2B).77,78 Unlike HCV NS3, flavi-virus helicases possess an RNA triphosphatase activity, As reviewed elsewhere in more detail,12,26,96 cells need heli- meaning they can cleave the terminal phosphate present at cases to evade the effect of drugs that kill cancer cells by dam- the 5′-end of RNA, to prepare the genome for capping.79 aging cancer cell DNA. For example, topoisomerase inhibitors, HCV genomes are not capped and HCV translation instead such as camptothecin, cause DNA strand breaks that need to be begins at an internal ribosome entry site (IRES).80 repaired by homologous recombination for a cell to survive or HCV infects about 1 in 50 people alive today, but most for a tumor cell to duplicate. Many of the human proteins HCV patients are unaware of their illness because the virus needed for recombinational DNA repair resemble the E. coli destroys the liver so slowly that it causes few symptoms.81 helicase RecQ protein, and helicase inhibitors that target After years of infection, most HCV patients develop fibro- human RecQ-like helicases could, in theory, make cancer cells sis, cirrhosis, hepatocellular carcinoma, or liver failure.82 more sensitive to chemotherapy.97,98 Effective HCV treatments combine pegylated human inter- Human RecQ-like helicases were first discovered when feron, ribavirin, and an inhibitor of the NS3/NS4A prote- some of their genes were found to be linked to various autoso- ase.83,84 The flaviviruses are primarily transmitted to mal recessive diseases, such as the premature aging disorder humans through mosquitoes, and they cause numerous mild Werner syndrome.28 There are five known human RecQ-like to fatal diseases. Vaccines are available for both YFV and proteins: RECQ1, WRN, BLM, RecQ4, and RecQ5. Mutations JEV, but currently there are no specific drugs available for in the WRN gene (RECQL2) cause Werner syndrome,99 and the treatment of flavivirus diseases.85 mutations in the BLM gene (RECQL3) cause Bloom syn- Coronaviruses (viruses in the family Coronaviridae) are drome.100 Mutations in the RecQ4 gene (RECQL4) have been also (+)RNA viruses, but they have genomes more than implicated in Rothmund-Thomson syndrome,101 Rapadilino Shadrick et al. syndrome,102 and Baller-Gerold syndrome.103 RecQ-like heli- ATP, which fuels helicase movements. ATP hydrolysis cases play key roles in DNA replication, recombination, and (ATPase) assays are typically easier to design and execute, repair; stabilize the replication fork; and are needed for telo- are less costly, and are simpler to perform in a high-throughput mere stability.52 As evidence that targeting RecQ-like helicases format. In addition, numerous commercial kits available, might aid chemotherapy, cells exposed to small hairpin RNA designed to monitor protein kinases,118 can be modified to (shRNA) targeting WRN are more sensitive to methylselenic detect helicase-catalyzed ATP hydrolysis. Strand separation acid,104 and cells with mutated WRN respond more slowly to (i.e., unwinding) assays require more sophisticated reagents, DNA damage from UV light or chemotherapeutic agents, such such as modified oligonucleotides, that are not needed in as the topoisomerase inhibitor camptothecin.105 RecQ5 also ATPase assays.
aids in the recovery of stalled replication forks after camptoth- Helicase-catalyzed ATP hydrolysis is measured by mon- ecin treatment.97 The RecQ-like proteins are not the only heli- itoring either the loss of ATP or the appearance of adenosine cases that could potentially serve as anticancer drug targets. diphosphate (ADP) or inorganic phosphate (Pi). Most Pi Many other helicases are needed to repair genomes, and they assays are based on the Fiske-SubbaRow method119 or more form complex networks with other key proteins, including sensitive ammonium molybdate reagents that incorporate tumor suppressors105 and oncoproteins.106 Mutations in these the dye malachite green.120,121 Colorimetric phosphate networks lead to synthetic lethality, suggesting that small- assays can be challenging to perform as screens because molecule inhibitors of such helicases might reproduce the either ATP must be removed or multiple reagents must be same phenotype.16 added in a precisely timed procedure. Proprietary colori- Another manner in which helicase inhibitors could be metric reagents such as Biomol Green reagent (Enzo Life used to treat cancers would be to halt the activity of a heli- Sciences, Farmingdale, NY) or the CytoPhos reagent case needed to express a specific oncogene. Protein synthe- (Cytoskeleton, Inc., Denver, CO)122 are more amenable to sis dysregulation is associated with many human cancers, HTS. Miyata et al.123 recently reported an interesting new and it might result from abnormal activity of RNA helicases variant of these classic phosphate assays that uses the dye needed for translation. The best-studied example is the quinaldine red. A quinaldine red–phosphate complex RNA helicase eIF4A, which prepares mRNA for ribosome absorbs light where many white assay plates emit when binding by unwinding secondary structures in the 5′ untrans- excited at 430 nm, so that white plate fluorescence decreases lated region (UTR). Cells exposed to an eIF4A inhibitor when quinaldine red forms a complex with phosphate and show reduced expression of an oncoprotein that has been molybdate. As discussed below, Seguin et al.124 used this linked to breast cancer, called Mucin 1.106 quinaldine red assay to discover new inhibitors of the SV40 Below we will discuss small molecules that inhibit the TAg helicase.
above helicases and how they were identified using high- The alternatives to detecting Pi in an ATPase assay are to throughput screening (HTS) techniques. Of course, other couple ATP hydrolysis to another reaction, detect ATP methods could be used to modulate helicase activity. For remaining, or detect ADP. The classic coupled ATPase example, RNA interference is the main technique used to assays link ATP hydrolysis to either nicotinamide adenine demonstrate that a helicase is a potential therapeutic target. dinucleotide (NADH) reduction via pyruvate kinase and RNA interference was used to show that HIV replication lactate dehydrogenase125 or methylthioguanosine (MESG) requires both DDX3107 and RNA helicase A,108 that human hydrolysis via purine nucleoside phosphorylase.122,126 liver cells need the RNA helicase p68 to support HCV rep- Neither coupled assay is particularly useful in HTS because lication,109 and that suppression of the Werner helicase many small molecules absorb in the same wavelengths as makes cells more sensitive to cancer chemotherapy.110–112 NADH and MESG. There are commercial assays, however, Viral helicases encoded as parts of polyproteins are obvi- that detect ATP and ADP through coupled luminescent reac- ously more difficult to selectively knock down using small tions (e.g., ADP glo; Promega, Madison, WI) or by using interfering RNA (siRNA), but they have been inhibited ADP sensors. ADP sensors use antibodies bound to a fluo- using other biological macromolecules. For example, HCV rescent ADP analogue, which can be displaced by native replication has been repressed using therapeutic antibod- ADP produced in a helicase-catalyzed reaction. ADP sensor ies113,114 and RNA aptamers directed against the NS3 assays, commonly referred to as "Transcreener" assays, can be monitored with fluorescence intensity, polarization, or time-resolved fluorescence resonance energy transfer and Typical High-Throughput Assays Used
are available from Bellbrook Labs (Madison, WI) or Cisbio to Identify Helicase Inhibitors
BioAssays (Marcoule, France).
With all these possibilities, and only a few published Most high-throughput assays used to identify helicase studies that directly compare various methods, choosing an inhibitors in compound collections either detect the ability ATPase-based helicase screen can be challenging. Our labo- of a helicase to separate DNA (or RNA) strands or cleave ratory prefers colorimetric ATPase assays for their low cost, Journal of Biomolecular Screening 18(7) precision, and simplicity,127 but other laboratories prefer other techniques. For example, Seguin et al.128 compared a commercial malachite green–based kit (BioAssays System, Hayward, CA) and the ADP Hunter kit (DiscoverRX, Freemont, CA) and reported that ADP Hunter kit was more sensitive and had a higher signal/background in assays with the SV40 helicase.
Helicase unwinding assays are performed as end-point assays or as continuous assays. The prototype helicase unwinding end-point assay measures the conversion of dou- ble-stranded DNA to single-stranded DNA using an iso- tope-labeled oligonucleotide. Such assays can be adapted to BILS 22 BS
BAY 57-1293
HTS using GeneClean Glassmilk (MP Biomedicals, Santa CID 3010846
CID 491941
Ana, CA) and filter plates129 or by using a scintillation prox- CID 11397521
imity assay (SPA), where a radiolabeled oligonucleotide is Figure 1. Herpes simplex virus (HSV) helicase/primase
captured with a biotin-labeled oligonucleotide, which then inhibitors. BILS 22 BS (CID 3010846), BAY 57-1293 (CID binds to a scintillant bead.130 Radioactive helicase end-point 491941), and ASP2151 (CID 11397521).
assays have also been done with a FlashPlate (PerkinElmer, Waltham, MA).131,132 Two unwinding end-point assays that do not use radioisotopes have been described. One uses electrochemiluminescence (ECL) and a substrate made by Proof of Concept: HSV Helicase/
attaching a DNA nucleotide to a ruthenium chelate, which is trapped by a biotin-labeled strand and streptavidin-coated magnetic beads that are detected by ECL.133 Another uses The inspiration for much of the research discussed here an enzyme-linked immunosorbent assay to detect displace- comes from the fact that helicase inhibitors already have ment of a digoxigenin (DIG)–labeled strand from a biotin- been demonstrated to be potent antiviral agents that rival labeled strand in a streptavidin-coated well plate.134 many of the drugs used to treat herpes infections.18,19 More Continuous helicase unwinding assays typically monitor than 10 years ago, Boehringer Ingelheim1 and Bayer2 discov- changes in Förster resonance energy transfer (FRET) ered anti-HSV drugs that target the UL5/8/52 complex. between "donor" and "acceptor" chromophores tethered to Boehringer Ingelheim identified their inhibitors, typified by complementary strands of DNA (or RNA).135–138 Continuous BILS 22BS (Fig. 1), by screening for compounds that inhibit
assays are simpler and often less costly than end-point helicase-catalyzed DNA strand separation. These aminothia- assays, but they are plagued by compound interference zolylphenyls inhibit primase-catalyzed RNA synthesis and because many library compounds absorb or emit light at helicase-catalyzed ATP hydrolysis in the presence of nucleic wavelengths that overlap those of the fluorophores being acids, but they do not inhibit helicase-catalyzed ATP hydroly- monitored. As an attempt to minimize compound interfer- sis in the absence of DNA. Interestingly, the BILS series sta- ence, similar assays have been developed that monitor bilizes a helicase/primase:DNA complex, possibly preventing either fluorescence polarization139 or time-resolved fluores- the primase recycling needed to initiate new Okazaki cence instead of fluorescence intensity.140 Our laboratory relies mainly on FRET-based assays in The Bayer compounds, in contrast, were discovered which one strand of a helicase substrate is made of a molec- using a cell-based high-throughput cell survival assay, not ular beacon (i.e., an oligonucleotide containing both a intended to find helicase inhibitors per se.2 The initial hit FRET donor and acceptor that can form a hairpin).141 In from a screen of 420,000 compounds (BAY 38-9489) was such a molecular beacon-based helicase assay (MBHA), optimized to a highly potent (IC = 12 nM) thiazole amide substrate fluorescence decreases when ATP activates the derivative called BAY 57-1293 (Fig. 1). Genetic analysis of
helicase. An MBHA has two advantages over other FRET- HSV resistant to BAY 57-1293 revealed that mutations in based assays. First, no oligonucleotide trap is required in UL5 or UL52 confer resistance to BAY 57-1293. BAY the reaction, making it simpler and less costly.11 Second, 57-1293 inhibits purified UL5/52-catalyzed ATP hydrolysis compounds that bind the helicase substrate can be detected in the presence of DNA (IC = 30 nM). In HSV-infected because they decrease substrate fluorescence in the absence guinea pigs143 and rabbits,144 BAY 57-1293 relieves symp- of the helicase (or ATP).24,142 As discussed below, DNA toms and prevents viral relapse, but resistance mutations are binding compounds are frequent, nonspecific hits in heli- common in both clinical and laboratory isolates of HSV- case assays, and identifying them early is critical for effi- 1.145 Some alleles (e.g., K356T in UL5) confer resistance to cient helicase inhibitor development.
both BAY 57-1293 and BILS 22 BS. However, some HSV Shadrick et al. alleles resistant to BAY 57-1293 (e.g., A899T in UL52) are Another polyphenyl helicase inhibitor was reported in a still sensitive to BILS 22 BS.146 modeling study designed to find compounds that bind the The first helicase inhibitor to show success in the clinic is RNA binding cleft of DDX3. Using docking, virtual screen- a herpes primase/helicase inhibitor called ASP2151 (ame- ing, and tests of the ability of hits to inhibit DDX3-catalyzed namevir; Fig. 1). ASP2151 is an oxadiazolylphenyl-containing
DNA unwinding or ATP hydrolysis, Radi et al.156 found a compound that inhibits purified UL5/8/52-catalyzed ATP potent N,N′-diarylurea (CID 29766776; Fig. 2B) DDX3
hydrolysis (IC = 78 nM), primer synthesis (IC < 30 nM), helicase inhibitor (IC = 5 µM), which also inhibits HIV-1 and DNA unwinding (IC < 100 nM). ASP2151 inhibits replication in cell-based assays (IC = 15 µM) without HSV in cell culture147 and guinea pig models.148 ASP2151 is detectable toxicity at 100 µM. CID 29766776 notably also effective against thymidine kinase–deficient HSV strains resembles the potent antibacterial and antifungal triclocar- resistant to acyclovir,149 and resistance to ASP2151 is 1000 ban (CID 7547).
times less common than seen for acyclovir.150 When adminis- Compounds similar to triclocarban (CID 7547) also tered to patients with genital herpes, ASP2151 significantly inhibit SV40 TAg. The Southern Research Specialized reduces the median time for lesion healing.151,152 Biocontainment Screening Center tested compounds in the NIH collection for their ability to inhibit SV40 TAg-catalyzed Inhibitors of Helicase-Catalyzed ATP
ATP hydrolysis (AID 1909). After examining their 2153 hits in dose-response assays (AID 1903), compound interference counterscreens (AID 2501), and cytotoxicity assays (AID The most obvious inhibitors of helicase-catalyzed ATP hydro- 2102), as well as examining the common features of the hits, lysis are nucleotide and nucleobase analogues. As reviewed the team discovered that Bisphenol A (BPA; CID 6623; Fig.
previously,8,153 nucleotide analogues have been extensively 2C) inhibits TAg with an IC value of 41 µM. BPA is used in
tested as inhibitors of the NS3 helicase, but few inhibit the many plastic consumer food containers, and as an estrogen enzyme with IC values less than 50 µM. More recently, syn- receptor agonist, it might present an environmental hazard. thesized new ring expanded nucleosides (REN) were tested if BPA inhibits TAg-dependent DNA replication (EC = 6 they inhibited HIV-1 replication by targeting the cellular heli- µM), but it is cytotoxic at similar concentrations.128 case DDX3. The most potent REN, CID 44586781, inhibits Remarkably similar chemotypes were also obtained in DDX3-catalyzed RNA unwinding, and it suppresses HIV-1 screens of other libraries with a different assay monitoring replication in T cells and macrophages.154 TAg-catalyzed ATP hydrolysis.124 For example, bithionol Other than nucleotides, the most common phamacoph- (CID 2406; Fig. 2C) and hexachlorophene (CID 3598), both
ores explored as inhibitors of helicase-catalyzed ATP hydro- of which are Food and Drug Administration–approved drugs, lysis are polyphenols made of two or three linked phenyl inhibit TAg.124 The bisphenol-like moiety, flexibility of the rings. Biphenyls have been studied as inhibitors of SV40 linker group, and the presence of substituents at positions 2 TAg,124,128 HPV E1,155 and DDX3,156 and triphenylmeth- and 4 on the phenols are all essential features needed for this anes have been twice studied as NS3 inhibitors.157,158 chemotype to inhibit SV40 TAg. Importantly, both bithionol Biphenyls were first noted as helicase inhibitors by and hexachlorophene inhibit SV40 and BKV cell culture, and researchers at Boehringer Ingelheim, who optimized this they are less toxic than BPA.124 chemotype as a lead to treat HPV. As previously Unpublished experiments in our laboratory have also reviewed,54,159 Boehringer Ingelheim tested its compound noted that some of the above biphenyls also inhibit the HCV collection for inhibitors of HPV E1-catalyzed ATP hydroly- helicase, but biphenyls are not as potent as triphenyl meth- sis, and the most promising screening hit was a biphenysul- anes known to inhibit NS3. Triphenylmethanes were first fonacetic acid (CID 515118; Fig. 2A), which inhibited
noted as NS3 inhibitors when the dye, soluble blue HT, was HPV6 E1-catalyzed ATP hydrolysis with an IC value of 2 found to dock in the ATP binding site and inhibit NS3 in µM (Fig. 2A).160 Compound optimization guided the dis-
assays with an IC value of 40 µM. A crystal structure covery of CID 515164 (Fig. 2A), which inhibits HPV6 E1
(PDB code 2ZJO) of blue HT bound to NS3 shows blue HT 500 times more potently than CID 515118. These biphenyl- in the ATP binding site, and it has been used to design CID sulfonacetic acids are reversible, but not linear competitive, 42618092 (Fig. 2D), a more potent triphenylmethane that
HPV helicase inhibitors, suggesting they bind an allosteric inhibits NS3 helicase and the HCV replicon.157 Mukherjee site, and they inhibit E1 isolated from some strains dramati- et al.158 found that a similar compound called aurintricar- cally better than E1 isolated from other HPV strains. boxylic acid (ATA; CID 2259; Fig. 2D) is an even more
Compound specificity results from the presence of tyrosine effective HCV helicase inhibitor, with an IC value of 1.4 at position 486 in HPV E1. When another residue is present µM. ATA also inhibits human RECQ1-catalyzed DNA in this position, the compounds bind more weakly. For unwinding (AID 2708) and the BLM helicase (AID 2528).
example, they are less active against HPV11 than they are Like other proteins with P-loop "Walker"-type ATP binding sites,161 magnesium forms a bridge needed for ATP Journal of Biomolecular Screening 18(7) to fuel helicase action.121 In theory, this bridge could be blocked by metal ion chelators, such as aryl diketoacids (ADKs). ADKs inhibit the unwinding activity of SARS- CoV helicase with IC values ranging from 5.4 to 13.6 µM. Dihydroxychromones, a class of naturally occurring flavo- CID 515118
noids, are bioisosteres of ADKs with better stability and safety. Dihydroxychromones containing arylmethyl groups, catechol groups, or both inhibit SARS-CoV helicase-catalyzed ATP hydrolysis and DNA unwinding. For example, CID CID 515164
45270979, which contains an arylmethyl and a catechol moiety on either side of a dihydroxychromone, inhibits B: Human DDX3
SARS-CoV helicase-catalyzed DNA unwinding (IC = 8.1 µM) but not ATP hydrolysis. When two arylmethyl groups are on either side of the pharmacophore (e.g., CID 56929932), the compound inhibits both Nsp13-catalyzed ATP hydrolysis (IC = 4 µM) and DNA unwinding (IC = CID 29766776
11 µM). CID 56929932 also inhibits HCV replication in C: SV40 TAg
cells (EC = 4 µM), but its antiviral effect against SARS- CoV has not yet been reported. Similar compounds with only one arylmethyl or catechol group do not inhibit the Bisphenol A (BPA)
Inhibitors of Helicase-Catalyzed
Nucleic Acid Separation
The main problem with targeting helicases through their ATP binding site is that the motor domains lining the ATP binding cleft are highly conserved.46,47,161 Helicase DNA (or RNA) binding sites are less similar, so in theory, compounds D: HCV NS3
binding in place of nucleic acids might be less promiscuous. However, small molecules targeting helicase nucleic acid binding sites have been hard to discover. To find compounds that directly target unwinding, most teams have focused on compounds that inhibit helicase-catalyzed unwinding but do not inhibit helicase-catalyzed ATP hydrolysis.23 One CID 42618092
problem with this approach is that a vast majority of com- pounds that inhibit unwinding do so by interacting with the nucleic acid substrate, not the enzyme itself. Examples include ethidium bromide, actinomycin D, 4′,6′-diamidino-2-phenylindole (DAPI), daunorubicin, distamycin, ellipti-cine, mitoxantrone, nalidixic acid, or netropsin, many of which have been studied as helicase inhibitors since the first studies were done with herpes UL9164 and the human RecQ- like proteins.165 Many DNA binding pharmacophores, such as anthracy- Aurintricarboxylic Acid (ATA)
clines, acridones, tropolones, and amidinoanthracyclines, have been optimized as HCV helicase inhibitors, and these Figure 2. Polyphenyl helicase inhibitors. (A) Inhibitors of
have been reviewed elsewhere.21,23 The inhibitory effects of the human papillomavirus (HPV) E1-catalyzed adenosine optimized acridones and tropolones on HCV helicase have triphosphate (ATP) hydrolysis: CID 515118, IC = 2 µM160; CID 515164, IC = 0.004 µM.155,160 (B) Human DDX3 inhibitors.
Fluoroquinolone antibiotics, which also bind nucleic acids, CID 29766776, IC = 5 µM.156 (C) Inhibitors of simian virus 40
have also been studied as inhibitors of SV40 TAg166 and the (SV40) TAg-catalyzed ATP hydrolysis: bisphenol A (CID 6623), IC = 41 µM128; bithionol (CID 2406), IC = 4 µM.124 (D)
Flavones comprise another pharmacophore with nucleic Hepatitis C virus (HCV) helicase inhibitors: CID 42618092, IC = 10 µM157; aurintricarboxylic acid (CID 2259), IC = 1.4 µM.158 acid binding capacity that has been frequently seen in Shadrick et al. screens for helicase inhibitors. For example, myricetin (CID that are linked head to tail rather than head to head. The sym- 5281672) and related flavones, such as luteolin and morin, metrical benzimidazoles inhibit HCV helicase by binding in all inhibit the hexameric replicative helicases, and myrice- place of RNA,177 but many retain an ability to interact with tin inhibits gram-negative bacteria growth, with a minimal nucleic acids,178 so they are rather promiscuous, inhibiting inhibitory concentration (MIC) as low as 0.25 mg/mL.168 NS3 from flaviviruses, and human DDX3.177 Myricetin (CID 5281672) and scutellarein (CID 5281697) Li et al.142 chose to study primuline because Belon and also inhibit SARS-CoV helicase with IC values of 2.7 µM Frick179 found a related dye called thioflavine S to be a hit and 0.9 µM, respectively.50,169 However, myricetin is also a in a small screen.179 To better understand how the dyes exert potent inhibitor of numerous DNA and RNA polymerases their action, Li et al. purified their active components and and telomerases,170 likely due to nonspecific interactions found that thioflavine S is composed of two major compo- with DNA or nucleic acid binding proteins.
nents. The related dye primuline is composed of two major Although some of the discussion above suggests that and four minor components, all of which are 1- to 4-unit-long helicases function nonspecifically on any duplex structure, benzothiazole oligomers terminating with a p-aminobenzene many helicases are known to act mainly on specific group.142 Their potency in helicase assays correlates with sequences or secondary structures such as hairpins, the length of the benzothiazole chain. All are reversible G-quadruplexes, or Holliday junctions.171 It might be pos- helicase inhibitors, and they inhibit NS3h by preventing the sible, therefore, to use small molecules that bind certain protein from binding single-stranded DNA or RNA.158 They sequences or mimic DNA structures to target specific heli- also have three undesirable properties. First, they bind cases needed in a disease pathway. For example, porphyrins nucleic acids, albeit with a lower affinity than with which that mimic a G-quadruplex inhibit the RecQ helicase,172 and they inhibit NS3h. Second, they displace unrelated proteins similar bismuth porphyrin complexes inhibit the SARS from single-stranded DNA or RNA. Third, they inhibit the NS3 protease even in the absence of the helicase domain, Optimization of helicase inhibitors that bind nucleic suggesting a nonspecific interaction with NS3.127 These acids is challenging because of the lack of HTS assays properties were minimized through synthetic diversifica- capable of detecting small molecule–DNA interactions. tion. All derivatives were tested for their ability to inhibit Most groups have relied on assays that monitor the ability HCV helicase, to bind DNA, and to displace E. coli single- of a small molecule to decrease the fluorescence of DNA stranded DNA binding protein from an oligonucleotide. stained with a fluorescent intercalator (e.g., ethidium bro- The most potent and specific compounds were tested for mide174 or thiazole orange175). Such fluorescent intercalator their ability to either inhibit NS3-catalyzed ATP hydrolysis displacement (FID) assays, however, do not detect all com- or peptide cleavage.127,180 The rationale for this extensive pounds that interact with DNA. For example, the Scripps counterscreening effort was that benzothiazoles such as Research Institute Molecular Screening Center tested those found in primuline could be promiscuous, acting 290,731 compounds in the NIH small-molecule collection nonspecifically. The most potent and specific helicase and found 487 hits (AID 1845), but later Li et al.142 found inhibitor synthesized is a 3-Cl benzoyl analogue synthe- that several of the compounds that did not test positive in sized from the primuline dimer (CID 50930730; Fig. 3).180
this ethidium bromide–based FID did, in fact, bind DNA. Li CID 50930730 inhibits NS3 helicase but does not interact et al. therefore developed a different DNA binding assay with DNA, affect the SSB-DNA interaction, or potently using SYBR green I, which can detect the interaction of a inhibit the NS3 ATPase or NS3 protease.
wider range of compounds with DNA, but there is still no To understand if primuline derivatives reach their target guarantee that all DNA binding compounds will affect the in cells, Ndjomou et al.127 exploited the fact that most retain fluorescence of a SYBR green I–stained DNA. In our labo- fluorescent properties similar to primuline.181 Most of the ratory, we therefore use an MBHA11 to simultaneously primuline derivatives absorb light near 360 nm and emit detect compounds that bind DNA and inhibit helicase light near 500 nm. The new compounds stain live cells har- boring subgenomic HCV replicons, and some derivatives decrease the amount of HCV RNA present in a hepatoma Polycyclic Aromatic Polymers as NS3 Inhibitors cell line with an enhanced ability to harbor HCV replicons. The primuline derivative that is the most potent HCV anti- Li et al.142 used the MBHA to design specific NS3 inhibitors viral is CID 50930749 (Fig. 3).127
from polycyclic aromatic polymers purified from the yellow Mukherjee et al.158 used a DNA binding assay to find dye primuline. The basic scaffold of the primuline deriva- other compounds that also prevent HCV from loading on tives is similar to the symmetrical benzothiazole polymers DNA. The two most effective compounds they found were (e.g., (BIP) B, CID 247520; Fig. 3) that were first noted to
titan yellow (CID 73217) and the polysulfonated naphtha- inhibit HCV helicase by ViroPharma,176–178 except that they lene suramin (CID 5361) (Fig. 3), which inhibit HCV heli-
are made from benzothiazoles (rather than benzimidazoles) case with IC of 12 µM and 4 µM, respectively. Both Journal of Biomolecular Screening 18(7) CID 247520
CID 50930730
CID 50930749
Titan yellow
CID 73217
Figure 3. Polcyclic polymers that inhibit the hepatitis C virus (HCV) NS3 helicase. (BIP) B, CID 247520, IC = 5 µM177; CID
50930730, IC = 2 µM142; CID 50930749, IC = 15 µM127; titan yellow (CID 73217), IC = 12 µM; suramin (CID 5361), IC = 4 suramin and titan yellow are not specific like the optimized inhibit P. aeruginosa DnaB-catalyzed DNA unwinding primuline derivatives. Suramin and titan yellow also pre- (AID 261721). The compounds do not prevent gram-nega- vent the E. coli single-stranded DNA binding protein from tive bacterial cell growth and are cytotoxic toward HeLa binding to DNA158; suramin inhibits the activity of human cells, but the most potent, CID 4041506 (Fig. 4A), inhibits
eIF4A182 and human RecQ-like proteins (AID 2549), and it the growth of gram-positive bacteria, such as S. aureus, prevents the RNA-induced silencing complex from loading with an MIC of 4 µg/mL.
In a more recent study, Aiello et al.185 tested 78,588 com- pounds from the Microbiotix (MBX) library for their ability to Antibacterial Agents Targeting DnaB inhibit B. anthracis DnaB, as well as 108,026 compounds in the National Screening Laboratory for the Regional Centers Several groups have searched large compound collections of Excellence in Biodefense and Emerging Infectious Disease for inhibitors of the bacterial replicative helicase, DnaB. (NSRB) collection for the ability to inhibit DNA unwinding For example, McKay et al.184 tested more than 230,000 catalyzed by S. aureus DnaC. The ICCB-Longwood/NSRB compounds and found a series of triaminotriazines that Screening Facility (Harvard Medical School) has deposited Shadrick et al. A: Bacterial DnaB-like
B: Human RecQ-like
OCID 227681
CID 4041506
CID 49852229
CID 1296013
C: SARS-CoV
CID 2807230
CID 53377571
Figure 4. New inhibitors of helicase-catalyzed DNA unwinding. (A) Inhibitors targeting bacterial DnaB-like helicases. CID 4041506,
IC = 5 µM184; CID 1296013, IC = 12 µM185; CID 53377571, IC = 1 µM. (B) Inhibitors targeting human RecQ-like helicase. CID
227681, IC (WRN) = 20 µM187; CID 49852229, IC (BLM) = 5 µM (AID 504662). (C) Inhibitor targeting severe acute respiratory
syndrome coronavirus (SARS-CoV) helicase. CID 2807230, IC = 5.7 µM.93 results for assays performed with the S. aureus helicase in the screen of 2000 compounds from the National Cancer Institute PubChem BioAsay (AID 485395). The new DnaB inhibitors Diversity Set. CID 227681 inhibits WRN helicase but does discovered in these campaigns include coumarins (5 com- not affect the activity of related RECQ1, E. coli RecQ, and pounds), benzothiazoles (2 compounds), rhodanines (4 com- DnaB under the same conditions. CID 227681 does not pounds), triazines (2 compounds), N-phenylpyrroles (2 appear to interact with DNA in FID assays, but when it is compounds), and three not easily classified compounds. The administered to cells, it induces double-stranded DNA most promising DnaB inhibitor in this set is an aminocouma- (dsDNA) breaks, apoptosis, replication fork stalling, and rin (CID 1296013; Fig. 4A) that inhibits growth of a variety of
mitotic checkpoint control, and it delays the cells in S-phase. gram-positive bacteria (MIC 5 µg/mL).185 This coumarin These NSC19630-induced cellular phenotypes have all been scaffold has since been further optimized, and the optimized shown to be WRN dependent, suggesting a "dominant nega- compound incorporates a biphenyl moiety reminiscent of the tive" mechanism of action.
phamacophore seen in SV40 and HPV inhibitors (CID More extensive screens have been performed with the 53377571; Fig. 4A).186
WRN (AID 651767), RECQ1 (AID 2549), and BLM (AID 2528) helicases, and data are available in the PubChem New Inhibitors of Human RecQ-like Helicases BioAssay. The NIH Chemical Genomics Center performed a quantitative high-throughput screen to measure IC val- Several high-throughput screens using unwinding assays ues for more than 250,000 compounds in assays with each have recently focused on finding inhibitors of human RecQ- of the three RecQ-like helicases. Hits in these screens like proteins, and as with the DnaB assays noted above, include compounds such as those discussed above, includ- much of the data are available on the PubChem BioAssay.
ing triphenylmethanes, biphenyls, DNA binding com- Aggarwal et al.187 first showed that RecQ-like helicase pounds, suramin, and anthracenediones. This project led to inhibitors could be valuable molecular probes when they the development of a potent, selective BLM inhibitor that characterized the effects of NSC19630 (CID 227681; Fig.
became NIH molecular probe ML216 (CID 49852229; Fig.
4B), which was identified as a potent WRN inhibitor in a
4B). ML216 inhibits BLM with an IC of 0.97 µM and
Journal of Biomolecular Screening 18(7) WRN with an IC value of 12 µM, but the compound has compounds. The J-domain of SV40 TAg stimulates the no effect against the related RECQ1 helicase. ML216 (CID ATPase of Hsp70, an important heat shock protein that 49852229) treatment sensitizes cells to aphidicolin, and helps protect cells from virus-induced stress. By testing ML216 inhibits the proliferation of only cells that express compound ability to inhibit TAg J-domain–stimulated Hsp70-catalyzed ATP hydrolysis, Wright et al.191 found that
MAL2-11B (CID 5461634; Fig. 5B) inhibits TAg-
New Inhibitors of the SARS-CoV Helicase stimulated Hsp70 ATPase activity, endogenous TAg ATPase activity, and SV40 replication in plaque assays by 4.5-fold Another recent example of a new helicase inhibitor discov- when tested at 100 µM. MAL2-11B also inhibits BK virus ered using an unwinding assay-based high-throughput DNA replication in human kidney cells by 90% when the screen was identified from the Maybridge Hitfinder chemi- cells are treated with 15 µM MAL2-11B.
cal library and is an inhibitor of the SARS-CoV helicase, Small molecules have also been reported that disrupt the CID 2807230 (Fig. 4C). CID 2807230 blocks the ability of
interaction of the NS3 helicase with the NS3 protease and nsp13 to unwind double-stranded RNA (IC = 5.7 µM) and an HCV structural protein called "core" (Fig. 5C). HCV
dsDNA (IC = 5.30 µM) but not the ATPase activity. CID core is a highly basic protein that helps pack the viral RNA 2807230 inhibits the SARS-CoV replication in cells with- genome in the virus capsid. Mousseau et al.192 designed an out apparent toxicity. CID 2807230 also inhibits the WRN AlphaScreen to detect the interaction between the NS3 heli- helicase (AID 651768), but it does not inhibit HCV heli- case and HCV core, and they used it to show that core pep- case, Dengue helicase, Moloney murine leukemia virus tides and an indoline alkaloid-type compound (called reverse transcriptase, or the E. coli DNA polymerase I, SL201; Fig. 5C) disrupt the core–NS3 helicase interaction.
Klenow fragment polymerase.189 SL201 also prevents core from forming dimers, suggesting that core dimers must form in order for core to bind NS3. Disrupting Helicase Interactions with
SL201 inhibits HCV virus production in cell culture. More recently, new compounds were found that bind the Key Partners
interface between the NS3 helicase and protease, so that The best example of an antiviral drug that disrupts a critical they lock the protein in a "closed" conformation where pep- helicase interaction affects the binding of HPV E1 to the tides cannot access the NS3 protease active site. The com- HPV E2 protein, which helps load the E1 helicase on the pounds, like the one shown in PDB file 4B75 (Fig. 5C), are
HPV origin of replication. E2 is a DNA binding protein that potent protease inhibitors (IC = 0.1 µM) and inhibit HCV regulates viral gene transcription, and E2 helps segregate replication (EC = 0.4 µM) without apparent toxicity in cell the HPV genome when host cells divide. The impressive culture.193 The effects of these new allosteric NS3 inhibitors development of E1-E2 interaction inhibitors has been on NS3-catalyzed RNA unwinding or ATP hydrolysis have recently reviewed.54,159 Briefly, Boehringer Ingelheim first not been reported. However, the compounds have no effect found compounds that prevent inhibitors of E1-E2 binding on NS3 lacking its helicase domain.
with an SPA using purified E2, E1, and radiolabeled HPV DNA. Since E2 binds DNA very tightly, most inhibitors Targeting Biological Functions of
reduce the signal in this assay by disrupting the E1-E2 inter- action.54 Structure-based design and further chemical opti-
mization led to CHEMBL1207308 (Fig. 5A), which is still
Helicase inhibitors identified using biochemical assays one of the most potent small-molecule helicase inhibitors often do not exert biological effects because they fail to (IC = 6 nM). Lower molecular weight, less complex enter cells, or they are unstable if they successfully enter E1-E2 interaction inhibitors were discovered using a radio- cells. To find helicase inhibitors with better pharmacologi- labeled CHEMBL1207308 analogue and an SPA to screen cal properties, several groups have designed assays that an expanded compound library.54 An intriguing hit in the depend on active helicase. An in vitro translation assay and later screen was a racemic mixture with a structure similar one example of a cell-based helicase assay suitable for HTS to repaglinide, a type 2 diabetes drug.190 Chemical optimi- are discussed below. They have been used successfully to zation led to CID 11330698 (Fig. 5A), which is a potent
find eIF4A and RecBCD inhibitors.
inhibitor of the E1-E2 interaction (IC = 20 nM). Since eIF4A is needed for cap-dependent translation, Unfortunately, CID 11330698 is rapidly metabolized and eIF4A inhibitors often block translation of capped RNA but lowers glucose levels by 6% when administered to rats at 1 not translation initiated from an IRES, like the one used to initiate HCV polyprotein synthesis. Inhibitors of either The interaction of the similar SF3 helicase from SV40 IRES-mediated or cap-dependent translation can therefore with a key partner has also been exploited to find antiviral be identified using bicistronic reporter vectors, such as one Shadrick et al. used the same assay to screen marine extracts for natural A: HPV E1-E2 Interaction
products that inhibit translation and found that hippurista- nol (CID 9981822; Fig. 6A) selectively inhibits eIF4A's
ability to bind RNA. Hippuristanol binds to amino acids near two conserved motifs in the C-terminal domain of eIF4A.195 Another translation inhibitor, pateamine A (CID 10053416; Fig. 6A), stimulates the rate at which eIF4A
cleaves ATP by enhancing the protein's affinity for RNA.196 Pateamine A induces eIF4A dimerization, thereby forcing eIF4A to engage in RNA binding and preventing it from participating in ribosome recruitment needed for transla-tion.197 Another eIF4A-dependent translation inhibitor, sil- vestrol (CID 21301152; Fig. 6A), enhances mouse
CID 11330698
lymphoma sensitivity to chemotherapy198 and blocks trans- B: SV40 TAg-HSP70 Interaction
lation of the MUC1-C oncoprotein.106 RecBCD also has a biological role that can be exploited to identify inhibitors of the helicase complex. RecBCD pre- vents phages lacking Chi sites in their DNA from infecting CID 5461634
E. coli. Amundsen et al.199 exploited the ability of RecBCD to protect against phage infection in a clever assay to find RecBCD inhibitors in 326,100 compounds in the NIH col- lection (AID 449731). They used T4 phage lacking the pro- tective gene 2 protein, which caps DNA to prevent RecBCD binding. Compounds inhibiting RecBCD therefore allow phage to lyse E. coli. To find possible drugs to treat H. pylori, the same system was used except that the host lacked C: HCV NS3
the E. coli RecBCD gene and instead contained the H. pylori RecBCD homolog, called AddAB (AID 435030), present. As a counterscreen, they added compounds to E. coli without phage, to identify compounds that simply act by killing cells (AID 449728). The most active and specific new RecBCD inhibitors include nine nitrofurans, one cya- nothiophene, one modified pyrimidopyridone, and one nitrothiazole. Two of the most potent of these RecBCD inhibitors are CID 1045135 and CID 2295461 (Fig. 6B). In
helicase assays, both compounds specifically inhibit RecBCD helicase, and CID 1045135 also inhibits the Figure 5. Compounds that disrupt the interactions of helicases
RecBCD nuclease.
with other proteins. (A) Inhibitors for the human papillomavirus
(HPV) E1-E2 interaction. CHEMBL1207308, IC = 0.006 µM206;
CID 11330698, IC = 0.02 µM.54 (B) Inhibitor of the simian
virus 40 (SV40) TAg-Hsp70 interaction. CID 5461634, IC = 20 µM.191 (C) Compounds targeting the interaction of hepatitis C
Despite the recent progress and numerous newly reported virus (HCV) helicase with the core protein (IC = 15 µM)192 and helicase inhibitors with promising properties, only a few the NS3 protease (IC = 0.1 µM).193 highly potent and specific helicase inhibitors have been developed, most of which target the HSV and HPV heli- where the cap-dependent reading frame encodes firefly cases. More work clearly needs to be done before helicase luciferase, and the IRES-expressed reading frame encodes inhibitors become a common drug class. Standard in vitro renilla luciferase. Both enzymes in this system produce helicase assays still yield few hits and are confounded by light but with different substrates, so they can be monitored compounds that act nonspecifically or that simply make simultaneously in the same assay. Using this system, Novac DNA more difficult to separate. Use of the molecular bea- et al.182 screened more than 90,000 compounds, identifying con-based helicase assays is helpful for identifying inhibi- many known translation inhibitors and nucleic acid binding tors that exert their effects by interacting with nucleic acids. ligands, as well as helicase inhibitors that do not interact However, improved methods to identify DNA binding with RNA, such as suramin (Fig. 3C). Bordeleau et al.194
agents in a high-throughput format are still needed.
Journal of Biomolecular Screening 18(7) A: Human eIF4A
B: Bacterial RecBCD-like
CID 10053416
CID 9981822
CID 21301152
CID 1045135
CID 2295461
Figure 6. Inhibitors found using assays monitoring the biological function of helicases. (A) Inhibitors of eIF4A-dependent translation:
pateamine (CID 10053416),196 hippuristanol (CID 9981822),194 and silvestrol (CID 21301152).198 (B) Inhibitors of RecBCD-dependent
bacteriophage defense. CID 1045135, IC = 2.5 µM; CID 2295461, IC = 16 µM.199
High-resolution structures of the best compounds described multidisciplinary approach that combines novel in vitro and above bound to their targets would also speed their develop- cell-based screening methods, structural biology, and ratio- ment and the design of more potent and specific inhibitors. nal design will be needed to design new antibiotics, antivi- Co-structures of a helicase-bound inhibitor guided the design rals, and anticancer drugs that function by targeting DNA or of a few of the above compounds, notably those that bind the RNA helicases.
HCV NS3 ATP binding site (PDB 2ZJO),157 the NS3 protease-helicase interface (PDB 2B75),193 and HPV E2 (PDB 1R6N).200 Similar co-structures with other compounds highlighted above should be possible to obtain, too, because many of the proteins We thank Frank Schoenen and Jennifer Golden (University of discussed above have already been crystallized and high-reso- Kansas) for helpful advice while preparing this review.
lution models are already available.
Even though few co-structures exist today, there is still a Declaration of Conflicting Interests
wealth of structural information for most of the targets dis- The authors declared no potential conflicts of interest with respect cussed here, and these data are underused in many drug dis- to the research, authorship, and/or publication of this article.
covery programs. Available structures could be used for virtual screening, docking, or compound optimization. Some of this work has been done already, but due to space The authors disclosed receipt of the following financial support limitations, we have not discussed it here in much detail. for the research, authorship, and/or publication of this article: This Examples of helicase inhibitors discovered through molec- work was supported by the NIH (RO1 AI088001) and a grant from ular modeling include compounds targeting the nucleic acid the UWM research foundation (RGI 101X219).
binding site,201 flavivirus NS3,202 and the human DDX3 ATP binding site.203,204 Similar work with other targets might prove fruitful.
1. Crute, J. J.; Grygon, C. A.; Hargrave, K. D.; Simoneau, B.; More studies also need to be done to examine the bio- Faucher, A. M.; Bolger, G.; Kibler, P.; Liuzzi, M.; Cordingley, logical implication of helicase inhibition and whether these M. G. Herpes Simplex Virus Helicase-Primase Inhibitors Are compounds are reaching their desired targets in cells. Active in Animal Models of Human Disease. Nat. Med. 2002,
Monitoring a helicase in a cell is a difficult problem to solve, but it is not impossible, as has been demonstrated 2. Kleymann, G.; Fischer, R.; Betz, U. A.; Hendrix, M.; Bender, W.; Schneider, U.; Handke, G.; Eckenberg, P.; Hewlett, G.; with cell-based RecBCD assays.199 Single-molecule enzy- Pevzner, V.; et al. New Helicase-Primase Inhibitors as Drug mology205 might help in this regard, and it might also open Candidates for the Treatment of Herpes Simplex Disease. up new frontiers for monitoring helicases either in cells or Nat. Med. 2002, 8, 392–398.
in cell-free extracts, which could be useful in screening, tar- 3. Wang, Y.; Xiao, J.; Suzek, T. O.; Zhang, J.; Wang, J.; Zhou, get identification, or compound optimization. Regardless, a Z.; Han, L.; Karapetyan, K.; Dracheva, S.; Shoemaker, B. Shadrick et al. A., et al. PubChem's BioAssay Database. Nucleic Acids Res. 23. Belon, C. A.; Frick, D. N. NS3 Helicase Inhibitors. In 2012, 40, D400–D412.
Hepatitis C: Antiviral Drug Discovery and Development; He, 4. Delagoutte, E.; von Hippel, P. H. Helicase Mechanisms and Y., Tan, S. L., Eds.; Caister Academic: Norfolk, UK, 2011;
the Coupling of Helicases within Macromolecular Machines. pp. 237–256.
Part I: Structures and Properties of Isolated Helicases. Q. Rev. 24. Hanson, A. M.; Hernandez, J. J.; Shadrick, W. R.; Frick, D. Biophys. 2002, 35, 431–478.
N. Identification and Analysis of Inhibitors Targeting the 5. Delagoutte, E.; von Hippel, P. H. Helicase Mechanisms and Hepatitis C Virus NS3 Helicase. Methods Enzymol. 2012,
the Coupling of Helicases within Macromolecular Machines. Part II: Integration of Helicases into Cellular Processes. Q. 25. Lescar, J.; Luo, D.; Xu, T.; Sampath, A.; Lim, S. P.; Canard, B.; Rev. Biophys. 2003, 36, 1–69.
Vasudevan, S. G. Towards the Design of Antiviral Inhibitors 6. Pyle, A. M. Translocation and Unwinding Mechanisms of against Flaviviruses: The Case for the Multifunctional NS3 RNA and DNA Helicases. Annu. Rev. Biophys. 2008, 37,
Protein from Dengue Virus as a Target. Antiviral Res. 2008,
7. Kwong, A. D.; Rao, B. G.; Jeang, K. T. Viral and Cellular 26. Chu, W. K.; Hickson, I. D. RecQ Helicases: Multifunctional RNA Helicases as Antiviral Targets. Nat. Rev. Drug Discov. Genome Caretakers. Nat. Rev. Cancer 2009, 9, 644–654.
2005, 4, 845–853.
27. Suhasini, A. N.; Brosh, R. M. J. DNA Helicases Associated 8. Frick, D. N.; Lam, A. M. Understanding Helicases as a Means with Genetic Instability, Cancer, and Aging. Adv. Exp. Med. of Virus Control. Curr. Pharm. Des. 2006, 12, 1315–1338.
Biol. 2013, 767, 123–144.
9. Singleton, M. R.; Dillingham, M. S.; Wigley, D. B. Structure 28. Suhasini, A. N.; Brosh, R. M. J. Disease-Causing Missense and Mechanism of Helicases and Nucleic Acid Translocases. Mutations in Human DNA Helicase Disorders. Mutat. Res., Annu. Rev. Biochem. 2007, 76, 23–50.
10. Bolton, E. E.; Wang, Y.; Thiessen, P. A.; Bryant, S. H. 29. Larsen, N. B.; Hickson, I. D. RecQ Helicases: Conserved PubChem: Integrated Platform of Small Molecules and Guardians of Genomic Integrity. Adv. Exp. Med. Biol. 2013,
Biological Activities. Annu. Rep. Comp. Chem. 2008, 4, 217–
30. Singleton, M. R.; Sawaya, M. R.; Ellenberger, T.; Wigley, 11. Belon, C. A.; Frick, D. N. Monitoring Helicase Activity with D. B. Crystal Structure of T7 Gene 4 Ring Helicase Indicates Molecular Beacons. BioTechniques 2008, 45, 433–440, 442.
a Mechanism for Sequential Hydrolysis of Nucleotides. Cell 12. Xi, X. G. Helicases as Antiviral and Anticancer Drug Targets. 2000, 101, 589–600.
Curr. Med. Chem. 2007, 14, 883–915.
31. Li, D.; Zhao, R.; Lilyestrom, W.; Gai, D.; Zhang, R.; 13. Frick, D. N. Helicases as Antiviral Drug Targets. Drug News DeCaprio, J. A.; Fanning, E.; Jochimiak, A.; Szakonyi, G.; Perspect. 2003, 16, 355–362.
Chen, X. S. Structure of the Replicative Helicase of the 14. van Brabant, A. J.; Stan, R.; Ellis, N. A. DNA Helicases, Oncoprotein SV40 Large Tumour Antigen. Nature 2003, 423,
Genomic Instability, and Human Genetic Disease. Annu. Rev. Genomics Hum. Genet. 2000, 1, 409–459.
32. Skordalakes, E.; Berger, J. M. Structural Insights into RNA- 15. Gupta, R.; Brosh, R. M. J. Helicases as Prospective Targets Dependent Ring Closure and ATPase Activation by the Rho for Anti-Cancer Therapy. Anticancer Agents Med. Chem. Termination Factor. Cell 2006, 127, 553–564.
2008, 8, 390–401.
33. Eoff, R. L.; Raney, K. D. Intermediates Revealed in the 16. Aggarwal, M.; Brosh, R. M. J. Hitting the Bull's Eye: Kinetic Mechanism for DNA Unwinding by a Monomeric Novel Directed Cancer Therapy through Helicase-Targeted Helicase. Nat. Struct. Mol. Biol. 2006, 13, 242–249.
Synthetic Lethality. J. Cell. Biochem. 2009, 106, 758–763.
34. Wong, I.; Chao, K. L.; Bujalowski, W.; Lohman, T. M. DNA- 17. Kleymann, G. New Antiviral Drugs That Target Herpesvirus Induced Dimerization of the Escherichia coli rep Helicase: Helicase Primase Enzymes. Herpes 2003, 10, 46–52.
Allosteric Effects of Single-Stranded and Duplex DNA. J. 18. Kleymann, G. Helicase Primase: Targeting the Achilles Heel Biol. Chem. 1992, 267, 7596–7610.
of Herpes Simplex Viruses. Antivir. Chem. Chemother. 2004,
35. Sikora, B.; Chen, Y.; Lichti, C. F.; Harrison, M. K.; Jennings, T. A.; Tang, Y.; Tackett, A. J.; Jordan, J. B.; Sakon, J.; 19. Field, H. J.; Mickleburgh, I. The Helicase-Primase Complex Cameron, C. E.; et al. Hepatitis C Virus NS3 Helicase Forms as a Target for Effective Herpesvirus Antivirals. Adv. Exp. Oligomeric Structures That Exhibit Optimal DNA Unwinding Med. Biol. 2013, 767, 145–159.
Activity In Vitro. J. Biol. Chem. 2008, 283, 11516–11525.
20. Bretner, M.; Najda, A.; Podwinska, R.; Baier, A.; Paruch, K.; 36. Singleton, M. R.; Dillingham, M. S.; Gaudier, M.; Lipniacki, A.; Piasek, A.; Borowski, P.; Kulikowski, T. Inhibitors Kowalczykowski, S. C.; Wigley, D. B. Crystal Structure of of the NTPase/Helicases of Hepatitis C and Related Flaviviridae RecBCD Enzyme Reveals a Machine for Processing DNA Viruses. Acta Pol. Pharm. 2004, 61(Suppl), 26–28.
Breaks. Nature 2004, 432, 187–193.
21. Frick, D. N. The Hepatitis C Virus NS3 Protein: A Model 37. Story, R. M.; Weber, I. T.; Steitz, T. A. The Structure of the E. RNA Helicase and Potential Drug Target. Curr. Issues Mol. coli recA Protein Monomer and Polymer. Nature 1992, 355,
Biol. 2007, 9, 1–20.
22. Belon, C. A.; Frick, D. N. Helicase Inhibitors as Specifically 38. Lohman, T. M.; Tomko, E. J.; Wu, C. G. Non-hexameric DNA Targeted Antiviral Therapy for Hepatitis C. Future Virol. Helicases and Translocases: Mechanisms and Regulation. 2009, 4, 277–293.
Nat. Rev. Mol. Cell Biol. 2008, 9, 391–401.
Journal of Biomolecular Screening 18(7) 39. Yao, N.; Hesson, T.; Cable, M.; Hong, Z.; Kwong, A. D.; Le, 58. Bereket, W.; Hemalatha, K.; Getenet, B.; Wondwossen, T.; H. V.; Weber, P. C. Structure of the Hepatitis C Virus RNA Solomon, A.; Zeynudin, A.; Kannan, S. Update on Bacterial Helicase Domain. Nat. Struct. Biol. 1997, 4, 463–467.
Nosocomial Infections. Eur. Rev. Med. Pharmacol. Sci. 2012,
40. Velankar, S. S.; Soultanas, P.; Dillingham, M. S.; Subramanya, H. S.; Wigley, D. B. Crystal Structures of Complexes of PcrA 59. Enright, M. C.; Robinson, D. A.; Randle, G.; Feil, E. J.; DNA Helicase with a DNA Substrate Indicate an Inchworm Grundmann, H.; Spratt, B. G. The Evolutionary History of Mechanism. Cell 1999, 97, 75–84.
Methicillin-Resistant Staphylococcus aureus (MRSA). Proc. 41. Soultanas, P.; Wigley, D. B. DNA Helicases: ‘Inching Natl. Acad. Sci. U. S. A. 2002, 99, 7687–7692.
Forward'. Curr. Opin. Struct. Biol. 2000, 10, 124–128.
60. Eggleston, A. K.; Rahim, N. A.; Kowalczykowski, S. C. A 42. Gu, M.; Rice, C. M. Three Conformational Snapshots Helicase Assay Based on the Displacement of Fluorescent, of the Hepatitis C Virus NS3 Helicase Reveal a Ratchet Nucleic Acid–Binding Ligands. Nucleic Acids Res. 1996, 24,
Translocation Mechanism. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 521–528.
61. Dillingham, M. S.; Spies, M.; Kowalczykowski, S. C. 43. Appleby, T. C.; Anderson, R.; Fedorova, O.; Pyle, A. M.; RecBCD Enzyme Is a Bipolar DNA Helicase. Nature 2003,
Wang, R.; Liu, X.; Brendza, K. M.; Somoza, J. R. Visualizing ATP-Dependent RNA Translocation by the NS3 Helicase 62. Anderson, D. G.; Kowalczykowski, S. C. The Translocating from HCV. J. Mol. Biol. 2011, 405, 1139–1153.
RecBCD Enzyme Stimulates Recombination by Directing 44. Morris, P. D.; Raney, K. D. DNA Helicases Displace RecA Protein onto ssDNA in a Chi-Regulated Manner. Cell Oligonucleotides. 1997, 90, 77–86.
Biochemistry 1999, 38, 5164–5171.
63. Court, R.; Cook, N.; Saikrishnan, K.; Wigley, D. The Crystal 45. Fischer, C. J.; Maluf, N. K.; Lohman, T. M. Mechanism of Structure of Lambda-Gam Protein Suggests a Model for ATP-Dependent Translocation of E.coli UvrD Monomers along RecBCD Inhibition. J. Mol. Biol. 2007, 371, 25–33.
Single-Stranded DNA. J. Mol. Biol. 2004, 344, 1287–1309.
64. Cromie, G. A. Phylogenetic Ubiquity and Shuffling of the 46. Gorbalenya, A. E.; Koonin, E. V. Helicases: Amino Acid Bacterial RecBCD and AddAB Recombination Complexes. Sequence Comparisons and Structure-Function Relationships. J. Bacteriol. 2009, 191, 5076–5084.
Curr. Opin. Struct. Biol. 1993, 3, 419–429.
65. Cernik, C.; Gallina, K.; Brodell, R. T. The Treatment of 47. Iyer, L. M.; Leipe, D. D.; Koonin, E. V.; Aravind, L. Herpes Simplex Infections: An Evidence-Based Review. Evolutionary History and Higher Order Classification of Arch. Intern. Med. 2008, 168, 1137–1144.
AAA+ ATPases. J. Struct. Biol. 2004, 146, 11–31.
66. Marintcheva, B.; Weller, S. K. A Tale of Two HSV-1 48. Gilhooly, N. S.; Gwynn, E. J.; Dillingham, M. S. Superfamily Helicases: Roles of Phage and Animal Virus Helicases in 1 Helicases. Front. Biosci. 2013, 5, 206–216.
DNA Replication and Recombination. Prog. Nucleic Acid 49. Byrd, A. K.; Raney, K. D. Superfamily 2 helicases. Front. Res. Mol. Biol. 2001, 70, 77–118.
Biosci. 2012, 17, 2070–2088.
67. Muylaert, I.; Tang, K. W.; Elias, P. Replication and 50. Keum, Y. S.; Jeong, Y. J. Development of Chemical Inhibitors Recombination of Herpes Simplex Virus DNA. J. Biol. Chem. of the SARS Coronavirus: Viral Helicase as a Potential 2011, 286, 15619–15624.
Target. Biochem. Pharmacol. 2012, 84, 1351–1358.
68. Hartman, E. A.; Huang, D. Update on PML: Lessons from 51. Henn, A.; Bradley, M. J.; De La Cruz, E. M. ATP Utilization the HIV Uninfected and New Insights in Pathogenesis and and RNA Conformational Rearrangement by DEAD-box Treatment. Curr. HIV/AIDS Rep. 2008, 5, 112–119.
Proteins. Annu. Rev. Biophys. 2012, 41, 247–267.
69. Fanning, E.; Knippers, R. Structure and Function of Simian 52. Bennett, R. J.; Keck, J. L. Structure and Function of RecQ DNA Virus 40 Large Tumor Antigen. Annu. Rev. Biochem. 1992,
Helicases. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 79–97.
61, 55–85.
53. Enemark, E. J.; Joshua-Tor, L. Mechanism of DNA 70. zur Hausen, H. Papillomaviruses and Cancer: From Basic Translocation in a Replicative Hexameric Helicase. Nature Studies to Clinical Application. Nat. Rev. Cancer 2002, 2,
2006, 442, 270–275.
54. White, P. W.; Faucher, A. M.; Goudreau, N. Small Molecule 71. Chène, P. NS3 Helicases as Drug Targets. RNA 2009, 2, 2–
Inhibitors of the Human Papillomavirus E1-E2 Interaction. Curr. Top. Microbiol. Immunol. 2011, 348, 61–88.
72. Wu, J.; Bera, A. K.; Kuhn, R. J.; Smith, J. L. Structure of 55. Itsathitphaisarn, O.; Wing, R. A.; Eliason, W. K.; Wang, the Flavivirus Helicase: Implications for Catalytic Activity, J.; Steitz, T. A. The Hexameric Helicase DnaB Adopts a Protein Interactions, and Proteolytic Processing. J. Virol. Nonplanar Conformation during Translocation. Cell 2012,
2005, 79, 10268–10277.
73. Mastrangelo, E.; Milani, M.; Bollati, M.; Selisko, B.; 56. Rajendran, S.; Jezewska, M. J.; Bujalowski, W. Multiple-Step Peyrane, F.; Pandini, V.; Sorrentino, G.; Canard, B.; Konarev, Kinetic Mechanism of DNA-Independent ATP Binding and P. V.; Svergun, D. I.; et al. Crystal Structure and Activity of Hydrolysis by Escherichia coli Replicative Helicase DnaB Kunjin Virus NS3 Helicase; Protease and Helicase Domain Protein: Quantitative Analysis Using the Rapid Quench-Flow Assembly in the Full Length NS3 Protein. J. Mol. Biol. 2007,
Method. J. Mol. Biol. 2000, 303, 773–795.
57. Kaper, J. B.; Nataro, J. P.; Mobley, H. L. T. Pathogenic 74. De Colibus, L.; Speroni, S.; Coutard, B.; Forrester, N. Escherichia coli. Nat. Rev. Microbiol. 2004, 2, 123–140.
L.; Gould, E.; Canard, B.; Mattevi, A. Purification and Shadrick et al. Crystallization of Kokobera Virus Helicase. Acta Crystallogr. 91. Sharma, A.; Awasthi, S.; Harrod, C. K.; Matlock, E. F.; Khan, Sect. F Struct. Biol. Cryst. Commun. 2007, 63, 193–195.
S.; Xu, L.; Chan, S.; Yang, H.; Thammavaram, C. K.; Rasor, 75. Tomei, L.; Failla, C.; Santolini, E.; De Francesco, R.; La Monica, R. A.; et al. The Werner Syndrome Helicase Is a Cofactor for N. NS3 is a Serine Protease Required for Processing of Hepatitis HIV-1 Long Terminal Repeat Transactivation and Retroviral C Virus Polyprotein. J. Virol. 1993, 67, 4017–4026.
Replication. J. Biol. Chem. 2007, 282, 12048–12057.
76. Yao, N.; Reichert, P.; Taremi, S. S.; Prosise, W. W.; Weber, P. 92. Yedavalli, V. S.; Neuveut, C.; Chi, Y. H.; Kleiman, L.; Jeang, C. Molecular Views of Viral Polyprotein Processing Revealed K. T. Requirement of DDX3 DEAD Box RNA Helicase for by the Crystal Structure of the Hepatitis C Virus Bifunctional HIV-1 Rev-RRE Export Function. Cell 2004, 119, 381–392.
Protease-Helicase. Structure 1999, 7, 1353–1363.
93. Fang, J.; Kubota, S.; Yang, B.; Zhou, N.; Zhang, H.; Godbout, 77. Luo, D.; Xu, T.; Hunke, C.; Gruber, G.; Vasudevan, S. G.; R.; Pomerantz, R. J. A DEAD Box Protein Facilitates HIV-1 Lescar, J. Crystal Structure of the NS3 Protease-Helicase Replication as a Cellular Co-factor of Rev. Virology 2004,
from Dengue Virus. J. Virol. 2008, 82, 173–183.
78. Assenberg, R.; Mastrangelo, E.; Walter, T. S.; Verma, A.; 94. Fang, J.; Acheampong, E.; Dave, R.; Wang, F.; Mukhtar, Milani, M.; Owens, R. J.; Stuart, D. I.; Grimes, J. M.; Mancini, M.; Pomerantz, R. J. The RNA Helicase DDX1 Is Involved E. J. Crystal Structure of a Novel Conformational State of the in Restricted HIV-1 Rev Function in Human Astrocytes. Flavivirus NS3 Protein: Implications for Polyprotein Processing Virology 2005, 336, 299–307.
and Viral Replication. J. Virol. 2009, 83, 12895–12906.
95. Ma, J.; Rong, L.; Zhou, Y.; Roy, B. B.; Lu, J.; Abrahamyan, 79. Wengler, G.; Wengler, G. The NS 3 Nonstructural Protein L.; Mouland, A. J.; Pan, Q.; Liang, C. The Requirement of of Flaviviruses Contains an RNA Triphosphatase Activity. the DEAD-Box Protein DDX24 for the Packaging of Human Virology 1993, 197, 265–273.
Immunodeficiency Virus Type 1 RNA. Virology 2008, 375,
80. Spahn, C. M.; Kieft, J. S.; Grassucci, R. A.; Penczek, P. A.; Zhou, K.; Doudna, J. A.; Frank, J. Hepatitis C Virus IRES 96. Sharma, S.; Doherty, K. M.; Brosh, R. M. J. DNA Helicases RNA-Induced Changes in the Conformation of the 40S as Targets for Anti-Cancer Drugs. Curr. Med. Chem. Ribosomal Subunit. Science 2001, 291, 1959–1962.
Anticancer Agents 2005, 5, 183–199.
81. Edlin, B. R. Perspective: Test and Treat This Silent Killer. 97. Hu, Y.; Lu, X.; Zhou, G.; Barnes, E. L.; Luo, G. Recql5 Plays Nature 2011, 474, S18–S19.
an Important Role in DNA Replication and Cell Survival after 82. McHutchison, J. G. Understanding Hepatitis C. Am. J. Manag. Camptothecin Treatment. Mol. Biol. Cell 2009, 20, 114–123.
Care 2004, 10, S21–S29.
98. Lu, X.; Lou, H.; Luo, G. A Blm-Recql5 Partnership in 83. Lange, C. M.; Sarrazin, C.; Zeuzem, S. Review Article: Replication Stress Response. J. Mol. Cell Biol. 2011, 3, 31–
Specifically Targeted Anti-viral Therapy for Hepatitis C—A New Era in Therapy. Aliment Pharmacol. Ther. 2010, 32,
99. Yu, C. E.; Oshima, J.; Fu, Y. H.; Wijsman, E. M.; Hisama, F.; Alisch, R.; Matthews, S.; Nakura, J.; Miki, T.; Ouais, S.; 84. Garber, K. Hepatitis C: Move Over Interferon. Nat. et al. Positional Cloning of the Werner's Syndrome Gene. Biotechnol. 2011, 29, 963–966.
Science 1996, 272, 258–262.
85. Mackenzie, J. S.; Gubler, D. J.; Petersen, L. R. Emerging 100. Gray, M. D.; Shen, J. C.; Kamath-Loeb, A. S.; Blank, A.; Flaviviruses: The Spread and Resurgence of Japanese Sopher, B. L.; Martin, G. M.; Oshima, J.; Loeb, L. A. The Encephalitis, West Nile and Dengue Viruses. Nat. Med. 2004,
Werner Syndrome Protein Is a DNA Helicase. Nat. Genet. 1997, 17, 100–103.
86. Al-Ahdal, M. N.; Al-Qahtani, A. A.; Rubino, S. Coronavirus 101. Kitao, S.; Shimamoto, A.; Goto, M.; Miller, R. W.; Smithson, Respiratory Illness in Saudi Arabia. J. Infect. Dev. Ctries. W. A.; Lindor, N. M.; Furuichi, Y. Mutations in RECQL4 2012, 6, 692–694.
Cause a Subset of Cases of Rothmund-Thomson Syndrome. 87. Ivanov, K. A.; Ziebuhr, J. Human Coronavirus 229E Nat. Genet. 1999, 22, 82–84.
Nonstructural Protein 13: Characterization of Duplex- 102. Kellermayer, R.; Siitonen, H. A.; Hadzsiev, K.; Kestila, Unwinding, Nucleoside Triphosphatase, and RNA M.; Kosztolanyi, G. A Patient with Rothmund-Thomson 5′-Triphosphatase Activities. J. Virol. 2004, 78, 7833–7838.
Syndrome and All Features of RAPADILINO. Arch. 88. Reddy, T. R.; Tang, H.; Xu, W.; Wong-Staal, F. Sam68, RNA Dermatol. 2005, 141, 617–620.
Helicase A and Tap Cooperate in the Post-transcriptional 103. Van Maldergem, L.; Siitonen, H. A.; Jalkh, N.; Chouery, E.; Regulation of Human Immunodeficiency Virus and Type D De Roy, M.; Delague, V.; Muenke, M.; Jabs, E. W.; Cai, J.; Retroviral mRNA. Oncogene 2000, 19, 3570–3575.
Wang, L. L.; et al. Revisiting the Craniosynostosis-Radial 89. Roy, B. B.; Hu, J.; Guo, X.; Russell, R. S.; Guo, F.; Kleiman, Ray Hypoplasia Association: Baller-Gerold Syndrome L.; Liang, C. Association of RNA Helicase A with Human Caused by Mutations in the RECQL4 Gene. J. Med. Genet. Immunodeficiency Virus Type 1 Particles. J. Biol. Chem. 2006, 43, 148–152.
2006, 281, 12625–12635.
104. Cheng, W. H.; Wu, R. T.; Wu, M.; Rocourt, C. R.; Carrillo, 90. Cocude, C.; Truong, M. J.; Billaut-Mulot, O.; Delsart, J. A.; Song, J.; Bohr, C. T.; Tzeng, T. J. Targeting Werner V.; Darcissac, E.; Capron, A.; Mouton, Y.; Bahr, G. M. Syndrome Protein Sensitizes U-2 OS Osteosarcoma Cells A Novel Cellular RNA Helicase, RH116, Differentially to Selenium-Induced DNA Damage Response and Necrotic Regulates Cell Growth, Programmed Cell Death and Human Death. Biochem. Biophys. Res. Commun. 2012, 420, 24–28.
Immunodeficiency Virus Type 1 Replication. J. Gen. Virol. 105. Blander, G.; Zalle, N.; Leal, J. F.; Bar-Or, R. L.; Yu, C. 2003, 84, 3215–3225.
E.; Oren, M. The Werner Syndrome Protein Contributes Journal of Biomolecular Screening 18(7) to Induction of p53 by DNA Damage. FASEB J. 2000, 14,
ATP: The Work of Fiske and SubbaRow. J. Biol. Chem. 2002, 277, 21e.
106. Jin, C.; Rajabi, H.; Rodrigo, C. M.; Porco, J. A. J.; Kufe, D. 120. Lanzetta, P. A.; Alvarez, L. J.; Reinach, P. S.; Candia, O. Targeting the eIF4A RNA Helicase Blocks Translation of A. An Improved Assay for Nanomole Amounts of Inorganic the MUC1-C Oncoprotein. Oncogene, in press.
Phosphate. Anal. Biochem. 1979, 100, 95–97.
107. Ishaq, M.; Hu, J.; Wu, X.; Fu, Q.; Yang, Y.; Liu, Q.; Guo, 121. Frick, D. N.; Banik, S.; Rypma, R. S. Role of Divalent Metal D. Knockdown of Cellular RNA Helicase DDX3 by Short Cations in ATP Hydrolysis Catalyzed by the Hepatitis C Hairpin RNAs Suppresses HIV-1 Viral Replication without Virus NS3 Helicase: Magnesium Provides a Bridge for ATP Inducing Apoptosis. Mol. Biotechnol. 2008, 39, 231–238.
to Fuel Unwinding. J. Mol. Biol. 2007, 365, 1017–1032.
108. Bolinger, C.; Sharma, A.; Singh, D.; Yu, L.; Boris-Lawrie, 122. Funk, C. J.; Davis, A. S.; Hopkins, J. A.; Middleton, K. M. K. RNA Helicase A Modulates Translation of HIV-1 and Development of High-Throughput Screens for Discovery Infectivity of Progeny Virions. Nucleic Acids Res. 2010, 38,
of Kinesin Adenosine Triphosphatase Modulators. Anal. Biochem. 2004, 329, 68–76.
109. Goh, P. Y.; Tan, Y. J.; Lim, S. P.; Tan, Y. H.; Lim, S. G.; 123. Miyata, Y.; Chang, L.; Bainor, A.; McQuade, T. J.; Walczak, Fuller-Pace, F.; Hong, W. Cellular RNA Helicase p68 C. P.; Zhang, Y.; Larsen, M. J.; Kirchhoff, P.; Gestwicki, J. Relocalization and Interaction with the Hepatitis C Virus E. High-Throughput Screen for Escherichia coli Heat Shock (HCV) NS5B Protein and the Potential Role of p68 in HCV Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume RNA Replication. J. Virol. 2004, 78, 5288–5298.
by Exploiting Energy Transfer. J. Biomol. Screen. 2010, 15,
110. Grandori, C.; Wu, K. J.; Fernandez, P.; Ngouenet, C.; Grim, J.; Clurman, B. E.; Moser, M. J.; Oshima, J.; Russell, D. W.; 124. Seguin, S. P.; Ireland, A. W.; Gupta, T.; Wright, C. M.; Swisshelm, K.; et al. Werner Syndrome Protein Limits MYC- Miyata, Y.; Wipf, P.; Pipas, J. M.; Gestwicki, J. E.; Brodsky, Induced Cellular Senescence. Genes Dev. 2003, 17, 1569–1574.
J. L. A Screen for Modulators of Large T Antigen's ATPase 111. Futami, K.; Takagi, M.; Shimamoto, A.; Sugimoto, M.; Activity Uncovers Novel Inhibitors of Simian Virus 40 and Furuichi, Y. Increased Chemotherapeutic Activity of BK Virus Replication. Antiviral Res. 2012, 96, 70–81.
Camptothecin in Cancer Cells by siRNA-Induced Silencing 125. Suzich, J. A.; Tamura, J. K.; Palmer-Hill, F.; Warrener, P.; of WRN Helicase. Biol. Pharm. Bull. 2007, 30, 1958–1961.
Grakoui, A.; Rice, C. M.; Feinstone, S. M.; Collett, M. S. 112. Masuda, K.; Banno, K.; Yanokura, M.; Tsuji, K.; Kobayashi, Hepatitis C Virus NS3 Protein Polynucleotide-Stimulated Y.; Kisu, I.; Ueki, A.; Yamagami, W.; Nomura, H.; Nucleoside Triphosphatase and Comparison with the Tominaga, E., et al. Association of Epigenetic Inactivation Related Pestivirus and Flavivirus Enzymes. J. Virol. 1993,
of the WRN Gene with Anticancer Drug Sensitivity in Cervical Cancer Cells. Oncol. Rep. 2012, 28, 1146–1152.
126. Rieger, C. E.; Lee, J.; Turnbull, J. L. A Continuous 113. Artsaenko, O.; Tessmann, K.; Sack, M.; Haussinger, D.; Spectrophotometric Assay for Aspartate Transcarbamylase Heintges, T. Abrogation of Hepatitis C Virus NS3 Helicase and ATPases. Anal. Biochem. 1997, 246, 86–95.
Enzymatic Activity by Recombinant Human Antibodies. J. 127. Ndjomou, J.; Kolli, R.; Mukherjee, S.; Shadrick, W. R.; Gen. Virol. 2003, 84, 2323–2332.
Hanson, A. M.; Sweeney, N. L.; Bartczak, D.; Li, K.; 114. Prabhu, R.; Khalap, N.; Burioni, R.; Clementi, M.; Garry, Frankowski, K. J.; Schoenen, F. J.; et al. Fluorescent R. F.; Dash, S. Inhibition of Hepatitis C Virus Nonstructural Primuline Derivatives Inhibit Hepatitis C Virus NS3- Protein, Helicase Activity, and Viral Replication by a Catalyzed RNA Unwinding, Peptide Hydrolysis and Viral Recombinant Human Antibody Clone. Am. J. Pathol. 2004,
Replicase Formation. Antiviral Res. 2012, 96, 245–255.
128. Seguin, S. P.; Evans, C. W.; Nebane-Akah, M.; McKellip, 115. Hwang, B.; Cho, J. S.; Yeo, H. J.; Kim, J. H.; Chung, K. M.; S.; Ananthan, S.; Tower, N. A.; Sosa, M.; Rasmussen, L.; Han, K.; Jang, S. K.; Lee, S. W. Isolation of Specific and White, E. L.; Maki, B. E.; et al. High-Throughput Screening High-Affinity RNA Aptamers against NS3 Helicase Domain Identifies a Bisphenol Inhibitor of SV40 Large T Antigen of Hepatitis C Virus. RNA 2004, 10, 1277–1290.
ATPase Activity. J. Biomol. Screen. 2012, 17, 194–203.
116. Umehara, T.; Fukuda, K.; Nishikawa, F.; Kohara, M.; 129. Sivaraja, M.; Giordano, H.; Peterson, M. G. High- Hasegawa, T.; Nishikawa, S. Rational Design of Dual- Throughput Screening Assay for Helicase Enzymes. Anal. Functional Aptamers That Inhibit the Protease and Helicase Biochem. 1998, 265, 22–27.
Activities of HCV NS3. J. Biochem. 2005, 137, 339–347.
130. Kyono, K.; Miyashiro, M.; Taguchi, I. Detection of Hepatitis 117. Fukuda, K.; Umehara, T.; Sekiya, S.; Kunio, K.; Hasegawa, C Virus Helicase Activity Using the Scintillation Proximity T.; Nishikawa, S. An RNA Ligand Inhibits Hepatitis C Virus Assay System. Anal. Biochem. 1998, 257, 120–126.
NS3 Protease and Helicase Activities. Biochem. Biophys. 131. Earnshaw, D. L.; Pope, A. J. FlashPlate Scintillation Res. Commun. 2004, 325, 670–675.
Proximity Assays for Characterization and Screening of 118. Koresawa, M.; Okabe, T. High-Throughput Screening DNA Polymerase, Primase, and Helicase Activities. J with Quantitation of ATP Consumption: A Universal Non- Biomol. Screen 2001, 6, 39–46.
Radioisotope, Homogeneous Assay for Protein Kinase. 132. Hicham Alaoui-Ismaili, M.; Gervais, C.; Brunette, S.; Assay Drug Dev. Technol. 2004, 2, 153–160.
Gouin, G.; Hamel, M.; Rando, R. F.; Bedard, J. A Novel 119. Simoni, R. D.; Hill, R. L.; Vaughan, M. The Determination High Throughput Screening Assay for HCV NS3 Helicase of Phosphorus and the Discovery of Phosphocreatine and Activity. Antiviral Res. 2000, 46, 181–193.
Shadrick et al. 133. Zhang, L.; Schwartz, G.; O'Donnell, M.; Harrison, between Two Helicase-Primase Inhibitors in Their Mode R. K. Development of a Novel Helicase Assay Using of Interaction with the Antiviral Target. J. Antimicrob. Electrochemiluminescence. Anal. Biochem. 2001, 293, 31–37.
Chemother. 2008, 61, 1044–1047.
134. Hsu, C. C.; Hwang, L. H.; Huang, Y. W.; Chi, W. K.; Chu, 147. Chono, K.; Katsumata, K.; Kontani, T.; Kobayashi, M.; Y. D.; Chen, D. S. An ELISA for RNA Helicase Activity: Sudo, K.; Yokota, T.; Konno, K.; Shimizu, Y.; Suzuki, H. Application as an Assay of the NS3 Helicase of Hepatitis C ASP2151, a Novel Helicase-Primase Inhibitor, Possesses Virus. Biochem. Biophys. Res. Commun. 1998, 253, 594–599.
Antiviral Activity against Varicella-Zoster Virus and Herpes 135. Houston, P.; Kodadek, T. Spectrophotometric Assay for Simplex Virus Types 1 and 2. J. Antimicrob. Chemother. Enzyme-Mediated Unwinding of Double-Stranded DNA. 2010, 65, 1733–1741.
Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 5471–5474.
148. Katsumata, K.; Chono, K.; Sudo, K.; Shimizu, Y.; Kontani, 136. Bjornson, K. P.; Amaratunga, M.; Moore, K. J.; Lohman, T.; Suzuki, H. Effect of ASP2151, a Herpesvirus Helicase- T. M. Single-Turnover Kinetics of Helicase-Catalyzed Primase Inhibitor, in a Guinea Pig Model of Genital Herpes. DNA Unwinding Monitored Continuously by Fluorescence Molecules 2011, 16, 7210–7223.
Energy Transfer. Biochemistry 1994, 33, 14306–14316.
149. Himaki, T.; Masui, Y.; Chono, K.; Daikoku, T.; Takemoto, 137. Boguszewska-Chachulska, A. M.; Krawczyk, M.; M.; Haixia, B.; Okuda, T.; Suzuki, H.; Shiraki, K. Efficacy of Stankiewicz, A.; Gozdek, A.; Haenni, A. L.; Strokovskaya, ASP2151, a Helicase-Primase Inhibitor, against Thymidine L. Direct Fluorometric Measurement of Hepatitis C Virus Kinase-Deficient Herpes Simplex Virus Type 2 Infection In Helicase Activity. FEBS Lett. 2004, 567, 253–258.
Vitro and In Vivo. Antiviral Res. 2012, 93, 301–304.
138. Tani, H.; Fujita, O.; Furuta, A.; Matsuda, Y.; Miyata, R.; 150. Chono, K.; Katsumata, K.; Kontani, T.; Shiraki, K.; Suzuki, Akimitsu, N.; Tanaka, J.; Tsuneda, S.; Sekiguchi, Y.; Noda, H. Characterization of Virus Strains Resistant to the Herpes N. Real-Time Monitoring of RNA Helicase Activity Using Virus Helicase-Primase Inhibitor ASP2151 (Amenamevir). Fluorescence Resonance Energy Transfer In Vitro. Biochem. Biochem. Pharmacol. 2012, 84, 459–467.
Biophys. Res. Commun. 2010, 393, 131–136.
151. Tyring, S.; Wald, A.; Zadeikis, N.; Dhadda, S.; Takenouchi, 139. Xu, H. Q.; Zhang, A. H.; Auclair, C.; Xi, X. G. Simultaneously K.; Rorig, R. ASP2151 for the Treatment of Genital Herpes: Monitoring DNA Binding and Helicase-Catalyzed DNA A Randomized, Double-Blind, Placebo- and Valacyclovir- Unwinding by Fluorescence Polarization. Nucleic Acids Res. Controlled, Dose-Finding Study. J. Infect. Dis. 2012, 205,
2003, 31, e70.
140. Earnshaw, D. L.; Moore, K. J.; Greenwood, C. J.; Djaballah, 152. Katsumata, K.; Weinberg, A.; Chono, K.; Takakura, S.; H.; Jurewicz, A. J.; Murray, K. J.; Pope, A. J. Time-Resolved Kontani, T.; Suzuki, H. Susceptibility of Herpes Simplex Fluorescence Energy Transfer DNA Helicase Assays for High Virus Isolated from Genital Herpes Lesions to ASP2151, Throughput Screening. J. Biomol. Screen. 1999, 4, 239–248.
a Novel Helicase-Primase Inhibitor. Antimicrob. Agents 141. Tyagi, S.; Kramer, F. R. Molecular Beacons: Probes That Chemother. 2012, 56, 3587–3591.
Fluoresce upon Hybridization. Nat. Biotechnol. 1996, 14,
153. Borowski, P.; Mueller, O.; Niebuhr, A.; Kalitzky, M.; Hwang, L. H.; Schmitz, H.; Siwecka, M. A.; Kulikowsk, T. ATP- 142. Li, K.; Frankowski, K. J.; Belon, C. A.; Neuenswander, B.; Binding Domain of NTPase/Helicase as a Target for Hepatitis Ndjomou, J.; Hanson, A. M.; Shanahan, M. A.; Schoenen, C Antiviral Therapy. Acta Biochim. Pol. 2000, 47, 173–180.
F. J.; Blagg, B. S.; Aube, J., et al. Optimization of Potent 154. Yedavalli, V. S.; Zhang, N.; Cai, H.; Zhang, P.; Starost, M. Hepatitis C Virus NS3 Helicase Inhibitors Isolated from the F.; Hosmane, R. S.; Jeang, K. T. Ring Expanded Nucleoside Yellow Dyes Thioflavine S and Primuline. J. Med. Chem. Analogues Inhibit RNA Helicase and Intracellular Human 2012, 55, 3319–3330.
Immunodeficiency Virus Type 1 Replication. J. Med. Chem. 143. Baumeister, J.; Fischer, R.; Eckenberg, P.; Henninger, K.; 2008, 51, 5043–5051.
Ruebsamen-Waigmann, H.; Kleymann, G. Superior Efficacy 155. White, P. W.; Faucher, A. M.; Massariol, M. J.; of Helicase-Primase Inhibitor BAY 57-1293 for Herpes Welchner, E.; Rancourt, J.; Cartier, M.; Archambault, Infection and Latency in the Guinea Pig Model of Human J. Biphenylsulfonacetic Acid Inhibitors of the Human Genital Herpes Disease. Antivir. Chem. Chemother. 2007,
Papillomavirus Type 6 E1 Helicase Inhibit ATP Hydrolysis 18, 35–48.
by an Allosteric Mechanism Involving Tyrosine 486. 144. Kaufman, H. E.; Varnell, E. D.; Gebhardt, B. M.; Thompson, Antimicrob. Agents Chemother. 2005, 49, 4834–4842.
H. W.; Atwal, E.; Rubsamen-Waigmann, H.; Kleymann, G. 156. Radi, M.; Falchi, F.; Garbelli, A.; Samuele, A.; Bernardo, Efficacy of a Helicase-Primase Inhibitor in Animal Models V.; Paolucci, S.; Baldanti, F.; Schenone, S.; Manetti, F.; of Ocular Herpes Simplex Virus Type 1 Infection. J. Ocul. Maga, G., et al. Discovery of the First Small Molecule Pharmacol. Ther. 2008, 24, 34–42.
Inhibitor of Human DDX3 Specifically Designed to Target 145. Biswas, S.; Smith, C.; Field, H. J. Detection of HSV-1 Variants the RNA Binding Site: Towards the Next Generation HIV-1 Highly Resistant to the Helicase-Primase Inhibitor BAY Inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 2094–2098.
57-1293 at High Frequency in 2 of 10 Recent Clinical Isolates 157. Chen, C. S.; Chiou, C. T.; Chen, G. S.; Chen, S. C.; Hu, C. of HSV-1. J. Antimicrob. Chemother. 2007, 60, 274–279.
Y.; Chi, W. K.; Chu, Y. D.; Hwang, L. H.; Chen, P. J.; Chen, 146. Biswas, S.; Kleymann, G.; Swift, M.; Tiley, L. S.; Lyall, J.; D. S., et al. Structure-Based Discovery of Triphenylmethane Aguirre-Hernandez, J.; Field, H. J. A Single Drug-Resistance Derivatives as Inhibitors of Hepatitis C Virus Helicase. J. Mutation in HSV-1 UL52 Primase Points to a Difference Med. Chem. 2009, 52, 2716–2723.
Journal of Biomolecular Screening 18(7) 158. Mukherjee, S.; Hanson, A. M.; Shadrick, W. R.; Ndjomou, 173. Yang, N.; Tanner, J. A.; Wang, Z.; Huang, J. D.; Zheng, J.; Sweeney, N. L.; Hernandez, J. J.; Bartczak, D.; Li, K.; B. J.; Zhu, N.; Sun, H. Inhibition of SARS Coronavirus Frankowski, K. J.; Heck, J. A., et al. Identification and Analysis Helicase by Bismuth Complexes. Chem. Commun. (Camb). of Hepatitis C Virus NS3 Helicase Inhibitors Using Nucleic 2007, 42, 4413–4415.
Acid Binding Assays. Nucleic Acids Res. 2012, 40, 8607–8621.
174. Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick, 159. D'Abramo, C. M.; Archambault, J. Small Molecule Inhibitors M. P. A Simple, High-Resolution Method for Establishing of Human Papillomavirus Protein-Protein Interactions. Open DNA Binding Affinity and Sequence Selectivity. J. Am. Virol. J. 2011, 5, 80–95.
Chem. Soc. 2001, 123, 5878–5891.
160. Faucher, A. M.; White, P. W.; Brochu, C.; Grand-Maitre, 175. Boger, D. L.; Tse, W. C. Thiazole Orange as the Fluorescent C.; Rancourt, J.; Fazal, G. Discovery of Small-Molecule Intercalator in a High Resolution Fid Assay for Determining Inhibitors of the ATPase Activity of Human Papillomavirus DNA Binding Affinity and Sequence Selectivity of Small E1 Helicase. J. Med. Chem. 2004, 47, 18–21.
Molecules. Bioorg. Med. Chem. 2001, 9, 2511–2518.
161. Walker, J. E.; Saraste, M.; Runswick, M. J.; Gay, N. J. 176. Phoon, C. W.; Ng, P. Y.; Ting, A. E.; Yeo, S. L.; Sim, M. Distantly Related Sequences in the Alpha- and Beta- M. Biological Evaluation of Hepatitis C Virus Helicase Subunits of ATP Synthase, Myosin, Kinases and Other ATP- Inhibitors. Bioorg. Med. Chem. Lett. 2001, 11, 1647–1650.
Requiring Enzymes and a Common Nucleotide Binding 177. Belon, C. A.; High, Y. D.; Lin, T. I.; Pauwels, F.; Frick, Fold. EMBO J. 1982, 1, 945–951.
D. N. Mechanism and Specificity of a Symmetrical 162. Lee, C.; Lee, J. M.; Lee, N. R.; Kim, D. E.; Jeong, Y. J.; Chong, Y. Investigation of the Pharmacophore Space of Biochemistry 2010, 49, 1822–1832.
Severe Acute Respiratory Syndrome Coronavirus (SARS- 178. Tunitskaya, V. L.; Mukovnya, A. V.; Ivanov, A. A.; Gromyko, CoV) NTPase/Helicase by Dihydroxychromone Derivatives. A. V.; Ivanov, A. V.; Streltsov, S. A.; Zhuze, A. L.; Kochetkov, Bioorg. Med. Chem. Lett. 2009, 19, 4538–4541.
S. N. Inhibition of the Helicase Activity of the HCV NS3 163. Kim, M. K.; Yu, M. S.; Park, H. R.; Kim, K. B.; Lee, C.; Protein by Symmetrical Dimeric Bis-benzimidazoles. Bioorg. Cho, S. Y.; Kang, J.; Yoon, H.; Kim, D. E.; Choo, H., et al. Med. Chem. Lett. 2011, 21, 5331–5335.
2,6-Bis-arylmethyloxy-5-hydroxychromones with Antiviral 179. Belon, C.; Frick, D. N. Thioflavin S Inhibits Hepatitis C Activity against Both Hepatitis C Virus (HCV) and SARS- Virus RNA Replication and the Viral Helicase with a Novel Associated Coronavirus (SCV). Eur. J. Med. Chem. 2011,
Mechanism. FASEB J. 2010, 24, lb202.
180. Li, K.; Frankowski, K. J.; Hanson, A. M.; Ndjomou, J.; 164. Kwok, Y.; Zhang, W.; Schroth, G. P.; Liang, C. H.; Alexi, Shanahan, M. A.; Mukherjee, S.; Shadrick, W. R.; Sweeney, N.; Bruice, T. W. Allosteric Interaction of Minor Groove N. L.; Belon, C. A.; Neuenswander, B., et al. Hepatitis C Binding Ligands with UL9-DNA Complexes. Biochemistry Virus NS3 Helicase Inhibitor Discovery. In Probe Reports 2001, 40, 12628–12638.
from the NIH Molecular Libraries Program, in press.
165. Brosh, R. M. J.; Karow, J. K.; White, E. J.; Shaw, N. D.; 181. Graham, R. K.; Caiger, P. Fluorescence Staining for the Hickson, I. D.; Bohr, V. A. Potent Inhibition of Werner and Determination of Cell Viability. Appl Microbiol 1969, 17,
Bloom Helicases by DNA Minor Groove Binding Drugs. Nucleic Acids Res. 2000, 28, 2420–2430.
182. Novac, O.; Guenier, A. S.; Pelletier, J. Inhibitors of Protein 166. Ali, S. H.; Chandraker, A.; DeCaprio, J. A. Inhibition of Synthesis Identified by a High Throughput Multiplexed Simian Virus 40 Large T Antigen Helicase Activity by Translation Screen. Nucleic Acids Res. 2004, 32, 902–915.
Fluoroquinolones. Antivir. Ther. 2007, 12, 1–6.
183. Tan, G. S.; Chiu, C. H.; Garchow, B. G.; Metzler, D.; 167. Khan, I. A.; Siddiqui, S.; Rehmani, S.; Kazmi, S. U.; Ali, S. Diamond, S. L.; Kiriakidou, M. Small Molecule Inhibition H. Fluoroquinolones Inhibit HCV by Targeting Its Helicase. of RISC Loading. ACS Chem. Biol. 2012, 7, 403–410.
Antivir. Ther. 2012, 17, 467–476.
184. McKay, G. A.; Reddy, R.; Arhin, F.; Belley, A.; Lehoux, 168. Xu, H.; Ziegelin, G.; Schroder, W.; Frank, J.; Ayora, S.; D.; Moeck, G.; Sarmiento, I.; Parr, T. R.; Gros, P.; Pelletier, Alonso, J. C.; Lanka, E.; Saenger, W. Flavones Inhibit the J., et al. Triaminotriazine DNA Helicase Inhibitors with Hexameric Replicative Helicase RepA. Nucleic Acids Res. Antibacterial Activity. Bioorg. Med. Chem. Lett. 2006, 16,
2001, 29, 5058–5066.
169. Yu, M. S.; Lee, J.; Lee, J. M.; Kim, Y.; Chin, Y. W.; Jee, 185. Aiello, D.; Barnes, M. H.; Biswas, E. E.; Biswas, S. B.; Gu, J. G.; Keum, Y. S.; Jeong, Y. J. Identification of Myricetin S.; Williams, J. D.; Bowlin, T. L.; Moir, D. T. Discovery, and Scutellarein as Novel Chemical Inhibitors of the SARS Characterization and Comparison of Inhibitors of Bacillus Coronavirus Helicase, nsP13. Bioorg. Med. Chem. Lett. anthracis and Staphylococcus aureus Replicative DNA 2012, 22, 4049–4054.
Helicases. Bioorg. Med. Chem. 2009, 17, 4466–4476.
170. Griep, M. A.; Blood, S.; Larson, M. A.; Koepsell, S. A.; Hinrichs, 186. Li, B.; Pai, R.; Di, M.; Aiello, D.; Barnes, M. H.; Butler, S. H. Myricetin Inhibits Escherichia coli DnaB Helicase but Not M. M.; Tashjian, T. F.; Peet, N. P.; Bowlin, T. L.; Moir, Primase. Bioorg. Med. Chem. 2007, 15, 7203–7208.
D. T. Coumarin-Based Inhibitors of Bacillus anthracis and 171. Sharma, S. Non-B DNA Secondary Structures and Their Staphylococcus aureus Replicative DNA Helicase: Chemical Resolution by RecQ Helicases. J. Nucleic Acids 2011, 2011,
Optimization, Biological Evaluation, and Antibacterial Activities. J. Med. Chem. 2012, 55, 10896–10908.
172. Wu, X.; Maizels, N. Substrate-Specific Inhibition of RecQ 187. Aggarwal, M.; Sommers, J. A.; Shoemaker, R. H.; Brosh, R. Helicase. Nucleic Acids Res. 2001, 29, 1765–1771.
M. J. Inhibition of Helicase Activity by a Small Molecule Shadrick et al. Impairs Werner Syndrome Helicase (WRN) Function in the 198. Bordeleau, M. E.; Robert, F.; Gerard, B.; Lindqvist, L.; Cellular Response to DNA Damage or Replication Stress. Chen, S. M.; Wendel, H. G.; Brem, B.; Greger, H.; Lowe, Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 1525–1530.
S. W.; Porco, J. A. J., et al. Therapeutic Suppression of 188. Nguyen, G. H.; Dexheimer, T. S.; Rosenthal, A. S.; Chu, W. Translation Initiation Modulates Chemosensitivity in a K.; Singh, D. K.; Mosedale, G.; Bachrati, C. Z.; Schultz, L.; Mouse Lymphoma Model. J. Clin. Invest. 2008, 118, 2651–
Sakurai, M.; Savitsky, P., et al. A Small Molecule Inhibitor of the BLM Helicase Modulates Chromosome Stability in 199. Amundsen, S. K.; Spicer, T.; Karabulut, A. C.; Londono, Human Cells. Chem. Biol. 2013, 20, 55–62.
L. M.; Eberhart, C.; Fernandez Vega, V.; Bannister, T. 189. Adedeji, A. O.; Singh, K.; Calcaterra, N. E.; Dediego, M. D.; Hodder, P.; Smith, G. R. Small-Molecule Inhibitors of L.; Enjuanes, L.; Weiss, S.; Sarafianos, S. G. Severe Acute Bacterial AddAB and RecBCD Helicase-Nuclease DNA Respiratory Syndrome Coronavirus Replication Inhibitor Repair Enzymes. ACS Chem. Biol. 2012, 7, 879–891.
That Interferes with the Nucleic Acid Unwinding of the 200. Wang, Y.; Coulombe, R.; Cameron, D. R.; Thauvette, Viral Helicase. Antimicrob. Agents Chemother. 2012, 56,
L.; Massariol, M. J.; Amon, L. M.; Fink, D.; Titolo, S.; Welchner, E.; Yoakim, C., et al. Crystal Structure of the E2 190. Grell, W.; Hurnaus, R.; Griss, G.; Sauter, R.; Rupprecht, Transactivation Domain of Human Papillomavirus Type 11 E.; Mark, M.; Luger, P.; Nar, H.; Wittneben, H.; Muller, Bound to a Protein Interaction Inhibitor. J. Biol. Chem. 2004,
P. Repaglinide and Related Hypoglycemic Benzoic Acid Derivatives. J. Med. Chem. 1998, 41, 5219–5246.
201. Kandil, S.; Biondaro, S.; Vlachakis, D.; Cummins, A. C.; 191. Wright, C. M.; Seguin, S. P.; Fewell, S. W.; Zhang, H.; Coluccia, A.; Berry, C.; Leyssen, P.; Neyts, J.; Brancale, A. Ishwad, C.; Vats, A.; Lingwood, C. A.; Wipf, P.; Fanning, E.; Discovery of a Novel HCV Helicase Inhibitor by a De Novo Pipas, J. M., et al. Inhibition of Simian Virus 40 Replication Drug Design Approach. Bioorg. Med. Chem. Lett. 2009, 19,
by Targeting the Molecular Chaperone Function and ATPase Activity of T Antigen. Virus Res. 2009, 141, 71–80.
202. Mastrangelo, E.; Pezzullo, M.; De Burghgraeve, T.; Kaptein, 192. Mousseau, G.; Kota, S.; Takahashi, V.; Frick, D. N.; S.; Pastorino, B.; Dallmeier, K.; de Lamballerie, X.; Neyts, Strosberg, A. D. Dimerization-Driven Interaction of J.; Hanson, A. M.; Frick, D. N., et al. Ivermectin is a Potent Hepatitis C Virus Core Protein with NS3 Helicase. J. Gen. Inhibitor of Flavivirus Replication Specifically Targeting Virol. 2011, 92, 101–111.
NS3 Helicase Activity: New Prospects for an Old Drug. J. 193. Saalau-Bethell, S. M.; Woodhead, A. J.; Chessari, G.; Carr, Antimicrob. Chemother. 2012, 67, 1884–1894.
M. G.; Coyle, J.; Graham, B.; Hiscock, S. D.; Murray, C. 203. Hogbom, M.; Collins, R.; van den Berg, S.; Jenvert, R. M.; W.; Pathuri, P.; Rich, S. J., et al. Discovery of an Allosteric Karlberg, T.; Kotenyova, T.; Flores, A.; Karlsson Hedestam, Mechanism for the Regulation of HCV NS3 Protein G. B.; Schiavone, L. H. Crystal Structure of Conserved Function. Nat. Chem. Biol. 2012, 8, 920–925.
Domains 1 and 2 of the Human DEAD-box Helicase 194. Bordeleau, M. E.; Mori, A.; Oberer, M.; Lindqvist, L.; DDX3X in Complex with the Mononucleotide AMP. J. Mol. Chard, L. S.; Higa, T.; Belsham, G. J.; Wagner, G.; Tanaka, Biol. 2007, 372, 150–159.
J.; Pelletier, J. Functional Characterization of IRESes by 204. Maga, G.; Falchi, F.; Garbelli, A.; Belfiore, A.; Witvrouw, an Inhibitor of the RNA Helicase eIF4A. Nat. Chem. Biol. M.; Manetti, F.; Botta, M. Pharmacophore Modeling and 2006, 2, 213–220.
Molecular Docking Led to the Discovery of Inhibitors of 195. Lindqvist, L.; Oberer, M.; Reibarkh, M.; Cencic, R.; Human Immunodeficiency Virus–1 Replication Targeting Bordeleau, M. E.; Vogt, E.; Marintchev, A.; Tanaka, J.; the Human Cellular Aspartic Acid–Glutamic Acid–Alanine– Fagotto, F.; Altmann, M., et al. Selective Pharmacological Aspartic Acid Box Polypeptide 3. J. Med. Chem. 2008, 51,
Targeting of a DEAD Box RNA Helicase. PLoS One 2008,
3, e1583.
205. Ha, T.; Kozlov, A. G.; Lohman, T. M. Single-Molecule 196. Bordeleau, M. E.; Matthews, J.; Wojnar, J. M.; Lindqvist, Views of Protein Movement on Single-Stranded DNA. L.; Novac, O.; Jankowsky, E.; Sonenberg, N.; Northcote, P.; Annu. Rev. Biophys. 2012, 41, 295–319.
Teesdale-Spittle, P.; Pelletier, J. Stimulation of Mammalian 206. Goudreau, N.; Cameron, D. R.; Deziel, R.; Hache, B.; Translation Initiation Factor eIF4A Activity by a Small Jakalian, A.; Malenfant, E.; Naud, J.; Ogilvie, W. W.; Molecule Inhibitor of Eukaryotic Translation. Proc. Natl. O'Meara, J.; White, P. W.; et al. Optimization and Acad. Sci. U. S. A. 2005, 102, 10460–10465.
Determination of the Absolute Configuration of a Series 197. Bordeleau, M. E.; Cencic, R.; Lindqvist, L.; Oberer, M.; of Potent Inhibitors of Human Papillomavirus Type-11 Northcote, P.; Wagner, G.; Pelletier, J. RNA-Mediated E1-E2 Protein-Protein Interaction: A Combined Medicinal Sequestration of the RNA Helicase eIF4A by Pateamine A Chemistry, NMR and Computational Chemistry Approach. Inhibits Translation Initiation. Chem. Biol. 2006, 13, 1287–1295.
Bioorg. Med. Chem. 2007, 15, 2690–2700.

Source: https://people.uwm.edu/frickd/files/2016/05/54_Shadrick-et-al-2013-1gwzqs0.pdf