Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

Facile Synthesis of Benzimidazoles via Oxidative Cyclization of Acyclic Monoterpene Aldehyde with Diamines: Studies on Antimicrobial and in Vivo Evaluation of Zebrafish

Citral ( 1a ), a bioactive component of Cymbopogon citratus (lemongrass) could be isolated and semi-synthetic analogs synthesized with improved therapeutic properties. Herein we first report describes citral ( 1a ) as a primary material for the synthesis of benzimidazole derivatives between various o-phenylenediamines ( 2a – l ) in the presence of Diisopropylethylamine (DIPEA) as a commercially available environmentally benign base, ethanol as a green solvent and the yield of all benzimidazole derivatives ( 3a – l ) was between 68–76 %; The semi-synthetically prepared benzimidazole derivatives ( 3a – l ) were assessed for their anti-bacterial and anti-fungal properties. The benzimidazole compounds ( 3a – b , and 3g – j ) exhibit good anti-microbial activity. In addition, in silico study was carried out to determine the specific binding affinity of the diamine halogen substituted benzimidazole derivatives to the specific target proteins. In silico analysis revealed a high correlation between docking results and experimental results. Finally, benzimidazole demonstrated significant antibacterial and antifungal activity. Zebrafish embryos were subjected to In vivo toxicological test found that all of the benzimidazole compounds ( 3a – l ) were non-toxic and had low embryotoxicity after 96 h, with an LC₅₀ of 36.425 μg, which could facilitate the design of novel antimicrobial agents using a cost-effective method.     

Three-dimensional solution structure of a unique S100 protein

S100A13 is a homodimeric protein that belongs to the S100 subfamily of EF-hand Ca2+-binding proteins. S100A13 exhibits unique physical and functional properties not observed in other members of the S100 family. S100A13 is crucial for the non-classical export of acidic fibroblast growth factors (FGFs-1), which lack signal peptide at their N-terminal end. In the present study, we report the three-dimensional solution structure of Ca2+-bound S100A13 using a variety of 3D NMR experiments. The structure of S100A13 is globular with four helices and an antiparallel beta-sheet in each subunit. The dimer interface is formed mainly by an antiparallel arrangement of helices H1, H1', H4, and H4'. Isothermal titration calorimetry (ITC) experiments show that S100A13 binds non-cooperatively to four calcium ions. Prominent differences exist between the three-dimensional structures of S100A13 and other S100 proteins. The hydrophobic pocket that largely contributes to protein-protein interactions in other S100 proteins is absent in S100A13. The structure of S100A13 is characterized by a large patch of negatively charged residues flanked by dense cationic clusters contributed largely by the positively charged residues located at the C-terminal end. Results of ITC experiments reveal that S100A13 lacking the C-terminal segment (residues 88-98) fails to bind FGF-1. The three-dimensional structure of S100A13 not only provides useful clues on its role in the non-classical export of signal peptide-less proteins such as FGF-1 but also paves the way for rational design of drugs against FGF-induced tumors.     

Human DNA helicase B (HDHB) binds to replication protein A and facilitates cellular recovery from replication stress

Maintenance of genomic stability in proliferating cells depends on a network of proteins that coordinate chromosomal replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of replication protein A (RPA) recruited to chromatin. We show using purified proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from replication stress.     

Cardiovascular effects of transdermally delivered bupranolol in rabbits: effect of chemical penetration enhancers

Bupranolol is a promising candidate for transdermal drug delivery system (TDDS) development. The effect of permeation enhancers on the in vivo delivery and beta-blocking effect of reservoir type TDDS was studied in comparison with intravenous BPL in rabbits. The beta-blocking effect was quantified by measuring the inhibition of isoprenaline induced tachycardia in rabbits after BPL administration via transdermal and intravenous routes. The reservoir type TDDS containing a hydroxypropyl cellulose gel and polyethylene membrane was used as a control device. In comparison, the TDDS containing skin penetration enhancers, either 2-pyrrolidone or partially methylated beta cyclodextrin (PMbetaCD) were evaluated. The control device (no enhancer) produced about 52% inhibition of isoprenaline induced tachycardia at 2 h and the effect continued over 24 h application period, however, the devices with 2-pyrolidone or PMbetaCD produced about 85% inhibition of isoprenaline induced tachycardia at 3 h and the same effect continued over 24 h application period. Likewise, the AUC of these devices were significantly higher than that of control device. The intravenous bupranolol showed rapid decline in the pharmacodynamic effect with time indicating its rapid elimination. The in vivo delivery of bupranolol (as estimated by a mass balance study) from the devices made with pyrolidone or PMbetaCD was 3-fold higher than that of control. The results of this study strongly suggest that the penetration enhancers in the TDDS increased the in vivo delivery of BPL, thereby increased the beta-blocking activity of BPL by 50-60% higher than control, enabling the reduction of the TDDS patch size, accordingly.     

The basic cleft of RPA70N binds multiple checkpoint proteins, including RAD9, to regulate ATR signaling

ATR kinase activation requires the recruitment of the ATR-ATRIP and RAD9-HUS1-RAD1 (9-1-1) checkpoint complexes to sites of DNA damage or replication stress. Replication protein A (RPA) bound to single-stranded DNA is at least part of the molecular recognition element that recruits these checkpoint complexes. We have found that the basic cleft of the RPA70 N-terminal oligonucleotide-oligosaccharide fold (OB-fold) domain is a key determinant of checkpoint activation. This protein-protein interaction surface is able to bind several checkpoint proteins, including ATRIP, RAD9, and MRE11. RAD9 binding to RPA is mediated by an acidic peptide within the C-terminal RAD9 tail that has sequence similarity to the primary RPA-binding surface in the checkpoint recruitment domain (CRD) of ATRIP. Mutation of the RAD9 CRD impairs its localization to sites of DNA damage or replication stress without perturbing its ability to form the 9-1-1 complex or bind the ATR activator TopBP1. Disruption of the RAD9-RPA interaction also impairs ATR signaling to CHK1 and causes hypersensitivity to both DNA damage and replication stress. Thus, the basic cleft of the RPA70 N-terminal OB-fold domain binds multiple checkpoint proteins, including RAD9, to promote ATR signaling.     

Inhibition of Herpes Simplex Virus Replication by a 2-Amino Thiazole via Interactions with the Helicase Component of the UL5-UL8-UL52 Complex

With the use of a high-throughput biochemical DNA helicase assay as a screen, T157602, a 2-amino thiazole compound, was identified as a specific inhibitor of herpes simplex virus (HSV) DNA replication. T157602 inhibited reversibly the helicase activity of the HSV UL5-UL8-UL52 (UL5/8/52) helicase-primase complex with an IC₅₀ (concentration of compound that yields 50% inhibition) of 5 μM. T157602 inhibited specifically the UL5/8/52 helicase and not several other helicases. The primase activity of the UL5/8/52 complex was also inhibited by T157602 (IC₅₀ = 20 μM). T157602 inhibited HSV growth in a one-step viral growth assay (IC₉₀ = 3 μM), and plaque formation was completely prevented at concentrations of 25 to 50 μM T157602. Vero, human foreskin fibroblast (HFF), and Jurkat cells could be propagated in the presence of T157602 at concentrations exceeding 100 μM with no obvious cytotoxic effects, indicating that the window between antiviral activity and cellular toxicity is at least 33-fold. Seven independently derived T157602-resistant mutant viruses (four HSV type 2 and three HSV type 1) carried single base pair mutations in the UL5 that resulted in single amino acid changes in the UL5 protein. Marker rescue experiments demonstrated that the UL5 gene from T157602-resistant viruses conferred resistance to T157602-sensitive wild-type viruses. Recombinant UL5/8/52 helicase-primase complex purified from baculoviruses expressing mutant UL5 protein showed complete resistance to T157602 in the in vitro helicase assay. T157602 and its analogs represent a novel class of specific and reversible anti-HSV agents eliciting their inhibitory effects on HSV replication by interacting with the UL5 helicase.Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) each comprise at least 77 genes whose expression is tightly regulated (42). These genes are assigned to four kinetic classes, designated as α, β, γ1, and γ2 on the basis of the timing of and requirements for their expression (46). The five α genes, α0, α4, α22, α27, and α47, are expressed first in the absence of viral protein synthesis and are responsible for the regulated expression of the other viral genes. The β genes require functional α gene products for their expression and encode proteins and enzymes that are directly involved in DNA synthesis and nucleotide metabolism. The γ genes form the last set of viral genes to be expressed, with the γ2 class having viral DNA replication as a strict requirement for their expression.The HSV genome contains three origins of replication (44, 45, 47, 48, 50, 54) and encodes seven viral proteins that are essential for DNA replication (34, 59). These include an origin binding protein (OBP) encoded by open reading frame (ORF) UL9 (14, 15, 17, 35), a DNA binding protein encoded by UL29 (40, 53, 54), a DNA polymerase encoded by ORF UL30 and its accessory factor encoded by UL42 (1, 4, 8, 18, 19, 21, 24, 37), and a heterotrimeric complex consisting of proteins encoded by ORFs UL5, UL8, and UL52, which include both 5′-to-3′ helicase activity and primase activity (10–12). Although extensively studied, the roles of the individual subunits of the helicase-primase complex and their specific interactions with each other have not been completely defined. However, several lines of evidence suggest that the UL5 gene encodes the helicase activity of the complex. Examination of the amino acid sequence of the UL5 protein revealed that it contains six conserved motifs that are found in many DNA and RNA helicases, two of these motifs defining an ATP binding site (20, 25, 32, 52, 61). Site-specific mutagenesis of amino acids within each of the six motifs revealed that all six are critical for the function of the UL5 protein as a helicase in transient replication assays (60, 61).The observation that recombinant UL5, UL52, and UL8 proteins could be purified from baculovirus-infected insect cells as a complex that displays DNA-dependent ATPase, helicase, and primase activities that are identical to those produced during a herpesvirus infection allowed functional and biochemical analyses of the individual components of the complex (10, 13, 38). Although the UL5 protein alone contained the defining helicase amino acid sequence motifs, the UL5 protein does not display helicase activity in vitro in the absence of the UL52 protein. Purified UL5 protein has less than 1% of the ATPase activity of the complex UL5-UL8-UL52 (UL5/8/52) complex (2, 43). In addition, studies with recombinant herpesviruses carrying mutations in the UL5 gene that abolish helicase activity revealed that the UL5 protein could still form specific interactions with UL8 and UL52 proteins (60). These results indicate that the functional domains of UL5 protein required for helicase activity are separate from those involved in protein-protein interactions and that UL5 and UL52 must interact to yield efficient helicase activity. Further mutagenesis studies with the UL52 protein identified mutations that abolish the primase activity of the complex, while the helicase and ATPase activities are unaffected, suggesting that the UL52 protein is responsible for the primase activity of the complex (27). The third component of the helicase-primase complex, the UL8 protein, interacts with other viral replication proteins, including the OBP, the single-stranded DNA binding protein, and the viral DNA polymerase (30, 33). It has been postulated that the interaction of the UL8 protein with the OBP (encoded by the UL9 gene) may function to recruit helicase-primase complexes to initiation complexes at viral origins (30). The UL8 protein is also required for stimulation of primer synthesis by the UL52 protein and for stimulation of the helicase activity of the helicase-primase complex which is crucial to allow efficient unwinding of long stretches of duplex DNA (16, 43, 49). Additionally, UL8 appears to be required for efficient nuclear entry of the helicase-primase complex (1, 3, 31).As the UL5, UL8, and UL52 gene products are essential for HSV replication and have not been exploited previously for antiviral drug discovery, they represent attractive targets for the development of novel anti-HSV agents. Current anti-HSV drugs include vidarabine (adenine arabinoside; Ara-A), foscarnet (phosphonoformic acid; PFA), and a wide variety of nucleoside analogs, the most clinically successful being acyclovir (ACV) and its analogs valacyclovir and famciclovir. ACV is phosphorylated by viral thymidine kinase (TK) to its monophosphate form, an event that occurs to a much lesser extent in uninfected cells. Subsequent phosphorylation events by cellular enzymes convert the ACV monophosphate to its triphosphate form. The ACV triphosphate derivative directly inhibits the DNA polymerase by competing as a substrate with dGTP. Because the ACV triphosphate lacks the 3′ hydroxyl group required to elongate the DNA chain, DNA replication is terminated. The triphosphorylated form of ACV is a much better substrate for the viral DNA polymerase than it is for the cellular DNA polymerase; thus, very little ACV triphosphate is incorporated into cellular DNA. Although ACV has proven to be safe and successful at reducing the duration, severity, and in some cases recurrence of HSV infections, eradication of the infection symptoms is far from complete and latent virus can reactivate frequently (55–58). In addition, primarily as a result of poor patient compliance with inconvenient ACV dosage regimens, virulent HSV strains resistant to ACV that contain mutations in either the viral TK or DNA polymerase gene have arisen (6, 7, 9, 26, 39). More potent and efficacious drugs that target other essential components of the virus replicative cycle would be invaluable as therapeutic agents to treat HSV and ACV-resistant HSV infections.To identify novel inhibitors of the HSV helicase-primase enzyme, we developed a high-throughput in vitro helicase assay and screened >190,000 samples. Using this biochemical approach, we identified T157602, a 2-amino thiazole, as a specific inhibitor of HSV replication. By generating and analyzing T157602-resistant viruses, we further demonstrate genetically that the molecular target of T157602 is the UL5 component of the HSV helicase-primase complex.