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.

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitorresistant influenza strains.

Typical assays used to discover and analyze small molecules that inhibit the hepatitis C virus (HCV) NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide binding assays are examined here as an alternative. After comparing fluorescence polarization (FP), homogeneous time-resolved fluorescence (HTRF®; Cisbio) and AlphaScreen® (Perkin Elmer) assays, an FP-based assay was chosen to screen Sigma’s Library of Pharmacologically Active Compounds (LOPAC) for compounds that inhibit NS3-DNA complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid (ATA) (IC 50 = 1.4 μM), suramin sodium salt (IC 50 = 3.6 μM), NF 023 hydrate (IC 50 = 6.2 μM) and tyrphostin AG 538 (IC 50 = 3.6 μM). All but AG 538 inhibited helicase-catalyzed strand separation, and all but NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli single-stranded DNA binding protein (SSB) revealed that none of the new HCV helicase inhibitors were specific for NS3h. However, when the SSB-based assay was used to analyze derivatives of another non-specific helicase inhibitor, the main component of the dye primuline, it revealed that some primuline derivatives (e.g. PubChem CID50930730) are up to 30-fold more specific for HCV NS3h than similarly potent HCV helicase inhibitors.

Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF 3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD-BeF 3 -trehalose and Tps2PD(D24N)-T6P complex structures reveal a "closed" conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate-protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD-BeF 3 -trehalose complex structure captures an aspartyl-BeF 3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an "open" conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102-103 and 184-188. Significantly, tps2Δ, tps2NTDΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens.trehalose-6-phosphate phosphatase | pathogenic fungi | HASDF phosphatase | trehalose-6-phosphate specificity | antifungal inhibitors

Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC values comparable to BET inhibitor (BETi) clinical candidates.

The hepatitis C virus (HCV) multifunctional nonstructural protein 3 (NS3) is a protease that cleaves viral and host proteins and a helicase that separates DNA and RNA structures in reactions fueled by ATP hydrolysis. Li et al. (J. Med. Chem. 2012, 55:3319-3330) recently synthesized a series of new NS3 helicase inhibitors from the benzothiazole dimer component of the fluorescent yellow dye primuline. This study further characterizes a subset of these primuline derivatives with respect to their specificity, mechanism of action, and effect on cells harboring HCV subgenomic replicons. All compounds inhibited DNA and RNA unwinding catalyzed by NS3 from different HCV genotypes, but only some inhibited the NS3 protease function, and few had any effect on HCV NS3 catalyzed ATP hydrolysis. A different subset were potent inhibitors of RNA stimulated ATP hydrolysis catalyzed by the related NS3 protein from Dengue virus. In assays monitoring intrinsic protein fluorescence in the absence of nucleic acids, the compounds cooperatively bound NS3 with Kd’s that reflect their potency in assays. The fluorescent properties of the primuline derivatives both in vitro and in cells are also described. The primuline derivative that was the most active against subgenomic replicons in cells caused a 14-fold drop in HCV RNA levels (IC50 = 5 ± 2 µM). In cells, the most effective primuline derivative did not inhibit the cellular activity of NS3 protease but disrupted HCV replicase structures.

Aurintricarboxylic acid (ATA) is a potent inhibitor of many enzymes needed for cell and virus replication, such as polymerases, helicases, nucleases, and topoisomerases. This study examines how ATA interacts with the helicase encoded by the hepatitis C virus (HCV) to reveal that ATA interferes with both nucleic acid and ATP binding to the enzyme. We show that ATA directly binds HCV helicase to prevent the enzyme from interacting with nucleic acids and to modulate the affinity of HCV helicase for ATP, the fuel for helicase action. Amino acid substitutions in the helicase DNA binding cleft or its ATP binding site alter the ability of ATA to disrupt helicase-DNA interactions. These data, along with molecular modeling results, support the notion that an ATA polymer binds between Arg467 and Glu493 to prevent the helicase from binding either ATP or nucleic acids. We also characterize how ATA affects the kinetics of helicase-catalyzed ATP hydrolysis, and thermodynamic parameters describing the direct interaction between HCV helicase and ATA using microcalorimetry. The thermodynamics of ATA binding to HCV helicase reveal that ATA binding does not mimic nucleic acid binding in that ATA binding is driven by a smaller enthalpy change and an increase in entropy.

This chapter describes two types of FRET-based fluorescence assays that can be used to identify and analyze compounds that inhibit the helicase encoded by the hepatitis C virus (HCV). Both assays use a fluorescently labeled DNA or RNA oligonucleotide to monitor helicase-catalyzed strand separation, and they differ from other real-time helicase assays in that they do not require the presence of other nucleic acids to trap the reaction products. The first assay is a molecular beacon-based helicase assay (MBHA) that monitors helicase-catalyzed displacement of a hairpin-forming oligonucleotide with a fluorescent moiety on one end and a quencher on the other. DNA-based MBHAs have been used extensively for high-throughput screening (HTS), but RNA-based MBHAs are typically less useful because of poor signal to background ratios. In the second assay discussed, the fluorophore and quencher are split between two hairpin-forming oligonucleotides annealed in tandem to a third oligonucleotide. This split beacon helicase assay can be used for HTS with either DNA or RNA oligonucleotides. These assays should be useful to the many labs searching for HCV helicase inhibitors in order to develop new HCV therapies that are still desperately needed.