Synthesis, Structural Determination, and Antioxidant Activities of Acyclic and Substituted Heterocyclic Phosphonates Linearly Linked 4-hydroxy-2(1H)-quinolinone.

The chemical reactivity of 3-[(E)-3-(dimethylamino)-2-propenoyl]-4-hydroxy-1-methy-2(1H)-quinolinone (1) towards some phosphorus reagents was studied. The enaminone 1 was cyclized into pyranoquinolinylphosphonate 2 via treatment with diethyl phosphite in basic medium. However, its reaction with triethoxy phosphonoacetate gave the substituted oxopyranylphosphonate 3. Using the same reaction conditions, both thioxopyridinylphosphonate 4 and oxopyranylphosphonate 5 were produced via a reaction of enaminone 1 with both diethyl 2-amino-2-thioxoethylphosphonate and diethyl vinylphosphonate, respectively, in low yields. In addition, the two novel oxopyridinylphosphonates 6 and 7 were obtained by treatment of enaminone 1 with a diethyl cyanomethylphosphonate reagent. Two oaxathiaphosphininyl derivatives, 8 and 9, were obtained by treatment of the enaminone 1 with O, O-diethyl dithiophosphoric acid under different reaction conditions. Diazaphosphininyl 11 and oxazaphosphininyl 12 derivatives were obtained in excellent yields using a P-phenylphosphonic diamide reagent under different reaction conditions. The treatment of the enaminone 1 with phosphorus pentasulfide produced the non-phosphorylated product thioxothiopyranoquinolinone 13. Finally, the enaminone was turned into oxathiaphosphininyl 14 using Lawesson's reagent. The possible reaction mechanisms of the formation of these products were discussed. The structures of newly isolated products were established by elemental analysis and spectral tools. The compounds were evaluated for their antioxidant activities.

Molecular docking of alpha-enolase to elucidate the promising candidates against Streptococcus pneumoniae infection.

PURPOSE: To predict potential inhibitors of alpha-enolase to reduce plasminogen binding of Streptococcus pneumoniae (S. pneumoniae) that may lead as an orally active drug. S. pneumoniae remains dominant in causing invasive diseases. Fibrinolytic pathway is a critical factor of S. pneumoniae to invade and progression of disease in the host body. Besides the low mass on the cell surface, alpha-enolase possesses significant plasminogen binding among all exposed proteins. METHODS: In-silico based drug designing approach was implemented for evaluating potential inhibitors against alpha-enolase based on their binding affinities, energy score and pharmacokinetics. Lipinski's rule of five (LRo5) and Egan's (Brain Or IntestinaL EstimateD) BOILED-Egg methods were executed to predict the best ligand for biological systems. RESULTS: Molecular docking analysis revealed, Sodium (1,5-dihydroxy-2-oxopyrrolidin-3-yl)-hydroxy-dioxidophosphanium (SF-2312) as a promising inhibitor that fabricates finest attractive charges and conventional hydrogen bonds with S. pneumoniae alpha-enolase. Moreover, the pharmacokinetics of SF-2312 predict it as a therapeutic inhibitor for clinical trials. Like SF-2312, phosphono-acetohydroxamate (PhAH) also constructed adequate interactions at the active site of alpha-enolase, but it predicted less favourable than SF-2312 based on binding affinity. CONCLUSION: Briefly, SF-2312 and PhAH ligands could inhibit the role of alpha-enolase to restrain plasminogen binding, invasion and progression of S. pneumoniae. As per our investigation and analysis, SF-2312 is the most potent naturally existing inhibitor of S. pneumoniae alpha-enolase in current time.

DNA Analogues Modified at the Nonlinking Positions of Phosphorus.

Chemically modified oligonucleotides are being developed as a new class of medicines for curing conditions that previously remained untreatable. Three primary classes of therapeutic oligonucleotides are single-stranded antisense oligonucleotides (ASOs), double stranded small interfering RNAs (siRNAs), and oligonucleotides that induce exon skipping. Recently, ASOs, siRNAs, and exon skipping oligonucleotides have been approved for patients with unmet medical needs, and many other candidates are being tested in late stage clinical trials. In coming years, therapeutic oligonucleotides may match the promise of small molecules and antibodies. Interestingly, in the 1980s when we developed chemical methods for synthesizing oligonucleotides, no one would have imagined that these highly charged macromolecules could become future medicines. Indeed, the anionic nature and poor metabolic stability of the natural phosphodiester backbone provided a major challenge for the use of oligonucleotides as therapeutic drugs. Thus, chemical modifications of oligonucleotides were essential in order to improve their pharmacokinetic properties. Keeping this view in mind, my laboratory has developed a series of novel oligonucleotides where one or both nonbridging oxygens in the phosphodiester backbone are replaced with an atom or molecule that introduces molecular properties that enhance biological activity. We followed two complementary approaches. One was the use of phosphoramidites that could act directly as synthons for the solid phase synthesis of oligonucleotide analogues. This approach sometimes was not feasible due to instability of various synthons toward the reagents used during synthesis of oligonucleotides. Therefore, using a complementary approach, we developed phosphoramidite synthons that can be incorporated into oligonucleotides with minimum changes in the solid phase DNA synthesis protocols but contain a handle for generating appropriate analogues postsynthetically.This Account summarizes our efforts toward preparing these types of analogues over the past three decades and discusses synthesis and properties of backbone modified oligonucleotides that originated from the Caruthers' laboratory. For example, by replacing one of the internucleotide oxygens with an acetate group, we obtained so-called phosphonoacetate oligonucleotides that were stable to nucleases and, when delivered as esters, entered into cells unaided. Alternatively oligonucleotides bearing borane phosphonate linkages were found to be RNase H active and compatible with the endogenous RNA induced silencing complex (RISC). Oligonucleotides containing an alkyne group directly linked to phosphorus in the backbone were prepared as well and used to attach molecules such as amino acids and peptides.

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment.

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ±â€¯0.1674 mM Pi and Vmax = 198.6 ±â€¯10.23 Pi × h-1 × mg protein-1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

Phosphorylation of Serine Induces Lysine p Ka Increases in Histone N-Termini and Signaling for Acetylation. Transcription Implications.

The mild acetylating agent, methyl acetyl phosphate, is used to estimate the p Ka values of some of the amine groups in peptides with sequences corresponding to a segment of the N-terminal tail of histone H4. When Ser-1 is not phosphorylated, the Lys ε amines have p Ka values in the range of 7.8-8.3, which are much lower than the currently assumed values. When Ser-1 is phosphorylated, the p Ka values of these Lys amines are elevated to the range of 8.8-10.3, thus providing the rationale for reports that they are then better substrates for acetyltransferases. Thus, reversal of suppressed p Ka values of Lys ε amines by Ser phosphorylation represents the basis for signaling in histone N-terminal tails to promote hyperacetylation, which is a hallmark of transcriptionally active euchromatin. In contrast, a state of hypoacetylation is present in the absence of phosphorylation as in transcriptionally inactive heterochromatin. A novel approach for estimating p Ka values based on a linkage between the Henderson-Hasselbalch and Michaelis-Menten equations indicates that the p Ka values of the Lys ε amines in H3 and H4 N-terminal tails have a highly variable charge gradient dependent on the location and proximity to the phosphorylation site.

Allostery and cooperativity in multimeric proteins: bond-to-bond propensities in ATCase.

Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. Second, cooperativity is explored by considering a bisubstrate analogue as the source of energy fluctuations at the active site, also leading to the identification of highly significant residues to the T ↔ R transition that enhance cooperativity across active sites. Finally, the inactive (T) structure is shown to exhibit a strong, non linear communication between the allosteric sites and the interface between catalytic subunits, rather than the active site. Bond-to-bond propensity thus offers an alternative route to explain allosteric and cooperative effects in terms of detailed atomistic changes to individual bonds within the protein, rather than through phenomenological, global thermodynamic arguments.

Mutant Cellular AP-1 Proteins Promote Expression of a Subset of Epstein-Barr Virus Late Genes in the Absence of Lytic Viral DNA Replication.

Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator.IMPORTANCE Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.

Pyrimidine synthesis inhibition enhances cutaneous defenses against antibiotic resistant bacteria through activation of NOD2 signaling.

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human ß-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.

Continuous DNA replication is required for late gene transcription and maintenance of replication compartments in gammaherpesviruses.

Late gene transcription in herpesviruses is dependent on viral DNA replication in cis but the mechanistic basis for this linkage remains unknown. DNA replication results in demethylated DNA, topological changes, removal of proteins and recruitment of proteins to promoters. One or more of these effects of DNA replication may facilitate late gene transcription. Using 5-azacytidine to promote demethylation of DNA, we demonstrate that late gene transcription cannot be rescued by DNA demethylation. Late gene transcription precedes significant increases in DNA copy number, indicating that increased template numbers also do not contribute to the linkage between replication and late gene transcription. By using serial, timed blockade of DNA replication and measurement of late gene mRNA accumulation, we demonstrate that late gene transcription requires ongoing DNA replication. Consistent with these findings, blocking DNA replication led to dissolution of DNA replication complexes which also contain RNA polymerase II and BGLF4, an EBV protein required for transcription of several late genes. These data indicate that ongoing DNA replication maintains integrity of a replication-transcription complex which is required for recruitment and retention of factors necessary for late gene transcription.

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines.

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06-0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24-128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6-10% of UTP left, and 12-36% CTP), followed by DCL and PF, which were almost similar (6-16% UTP and 12-27% CTP), while PALA was the least active compound (10-70% UTP and 13-68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31-72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools.

Etching Efficacy of Self-Etching Functional Monomers.

Besides chemically interacting with hard tooth tissue, acidic functional monomers of self-etch adhesives should etch the prepared tooth surface to dissolve the smear layer and to provide surface micro-retention. Although the etching efficacy of functional monomers is commonly determined in terms of pH, the pH of adhesives cannot accurately be measured. Better is to measure the hydroxyapatite (HAp)-dissolving capacity, also considering that functional monomers may form monomer-Ca salts. Here, the etching efficacy of 6 functional monomers (GPDM, phenyl-P, MTEGP, 4-META, 6-MHP and 10-MDP) was investigated. Solutions containing 15 wt% monomer, 45 wt% ethanol, and 40 wt% water were prepared. Initially, we observed enamel surfaces exposed to monomer solution by scanning electron microscopy (SEM). X-ray diffraction (XRD) was employed to detect monomer-Ca salt formation. Phenyl-P exhibited a strong etching effect, while 10-MDP-treated enamel showed substance deposition, which was identified by XRD as 10-MDP-Ca salt. To confirm these SEM/XRD findings, we determined the etching efficacy of functional monomers by measuring both the concentration of Ca released from HAp using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and the amount of monomer-Ca salt formation using 31P magic-angle spinning (MAS) nuclear magnetic resonance (NMR). ICP-AES revealed that the highest Ca concentration was produced by phenyl-P and the lowest Ca concentration, almost equally, by 4-META and 10-MDP. Only 10-MDP formed 10-MDP-Ca salts, indicating that 10-MDP released more Ca from HAp than was measured by ICP-AES. Part of the released Ca was consumed to form 10-MDP-Ca salts. It is concluded that the repeatedly reported higher bonding effectiveness of 10-MDP-based adhesives must not only be attributed to the more intense chemical bonding of 10-MDP but also to its higher etching potential, a combination the other functional monomers investigated lack.

PMIDA-Modified Fe3O4 Magnetic Nanoparticles: Synthesis and Application for Liver MRI.

The surface modification of Fe3O4-based magnetic nanoparticles (MNPs) with N-(phosphonomethyl)iminodiacetic acid (PMIDA) was studied, and the possibility of their use as magnetic resonance imaging contrast agents was shown. The effect of the added PMIDA amount, the reaction temperature and time on the degree of immobilization of this reagent on MNPs, and the hydrodynamic characteristics of their aqueous colloidal solutions have been systematically investigated for the first time. It has been shown that the optimum condition for the modification of MNPs is the reaction at 40 °C with an equimolar amount of PMIDA for 3.5 h. The modified MNPs were characterized by X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric, and CHN elemental analyses. The dependence of the hydrodynamic characteristics of the MNP colloidal solutions on the concentration and pH of the medium was studied by the dynamic light scattering method. On the basis of the obtained data, we can assume that the PMIDA molecules are fixed on the surface of the MNPs as a monomolecular layer. The modified MNPs had good colloidal stability and high magnetic properties. The calculated relaxivities r2 and r1 were 341 and 102 mmol-1 s-1, respectively. The possibility of using colloidal solutions of PMIDA-modified MNPs as a T2 contrast agent for liver studies in vivo (at a dose of 0.6 mg kg-1) was demonstrated for the first time.

Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition.

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.

Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs.

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.

Interferon-γ-inducible protein 16 (IFI16) is required for the maintenance of Epstein-Barr virus latency.

BACKGROUND: Epstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. It's during the latent phase of EBV that all EBV-associated cancers, including Burkitt's lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease arise. Interferon-γ-inducible protein 16 (IFI16) is a well-established innate immune sensor and viral transcriptional regulator involved in response to invading DNA viruses. During latency, IFI16 remains in the nucleus, in part bound to the EBV genome; however, neither its role in EBV lytic cycle or latency has been established. METHODS: Short interfering RNA against IFI16 and IFI16 overexpression were used to identify the role of IFI16 in the maintenance of EBV latency I. We also studied how induction of the lytic cycle affected IFI16 using the EBV positive, latently infected Akata or MUTU-1 cell lines. Akata cells were induced with TPA and MUTU-1 cells with TGF-ß up to 96 h and changes in IFI16 protein were analyzed by Western blotting and immunofluorescence microscopy. To assess the mechanism of IFI16 decrease, EBV DNA replication and late lytic transcripts were blocked using the viral DNA polymerase inhibitor phosphonoacetic acid. RESULTS: Knockdown of IFI16 mRNA by siRNA resulted in enhanced levels of EBV lytic gene expression from all temporal gene classes, as well as an increase in the total EBV genome abundance, whereas overexpression of exogenous IFI16 reversed these effects. Furthermore, 96 h after induction of the lytic cycle with either TPA (Akata) or TGF-ß (MUTU-1), IFI16 protein levels decreased up to 80% as compared to the EBV-negative cell line BJAB. Reduction in IFI16 was observed in cells expressing EBV lytic envelope glycoprotein. The decreased levels of IFI16 protein do not appear to be dependent on late lytic transcripts of EBV but suggest involvement of the immediate early, early, or a combination of both gene classes. CONCLUSIONS: Reduction of IFI16 protein levels following lytic cycle induction, as well as reactivation from latency after IFI16 mRNA knockdown suggests that IFI16 is crucial for the maintenance of EBV latency. More importantly, these results identify IFI16 as a unique host factor protein involved in the EBV lifecycle, making it a potential therapeutic target to combat EBV-related malignancies.

New Insights into DNA Polymerase Function Revealed by Phosphonoacetic Acid-Sensitive T4 DNA Polymerases.

The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments.

Conformational dynamics of 1-deoxy-d-xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS.

The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length Escherichia coli DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. (i) The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. (ii) Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. (iii) The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.

A Salmonella Regulator Modulates Intestinal Colonization and Use of Phosphonoacetic Acid.

Many microorganisms produce phosphonates, molecules characterized by stable carbon-phosphorus bonds that store phosphorus or act as antimicrobials. The role of phosphonates in the marine biosphere is well characterized but the role of these molecules in the intestine is poorly understood. Salmonella enterica uses its virulence factors to influence the host immune response to compete with the host and normal microflora for nutrients. Salmonella cannot produce phosphonates but encodes the enzymes to use them suggesting that it is exposed to phosphonates during its life cycle. The role of phosphonates during enteric salmonellosis is unexplored. We have previously shown that STM3602, encoding a putative regulator of phosphonate metabolism, is needed for colonization in calves. Here, we report that the necessity of STM3602 in colonization of the murine intestine results from multiple factors. STM3602 is needed for full activation of the type-3 secretion system-1 and for optimal invasion of epithelial cells. The ΔSTM3602 mutant grows poorly in phosphonoacetic acid (PA) as the sole phosphorus source, but can use 2-aminoethylphosphonate. PhnA, an enzyme required for PA breakdown, is not controlled by STM3602 suggesting an additional mechanism for utilization of PA in S. Typhimurium. Finally, the requirement of STM3602 for intestinal colonization differs depending on the composition of the microflora. Our data suggest that STM3602 has multiple regulatory targets that are necessary for survival within the microbial community in the intestine. Determination of the members of the STM3602 regulon may illuminate new pathways needed for colonization of the host.

RSDL decontamination of human skin contaminated with the nerve agent VX.

Dermal exposure to low volatile organophosphorus compounds (OPC) may lead to penetration through the skin and uptake in the blood circulation. Skin decontamination of toxic OPCs, such as pesticides and chemical warfare nerve agents, might therefore be crucial for mitigating the systemic toxicity following dermal exposure. Reactive skin decontamination lotion (RSDL) has been shown to reduce toxic effects in animals dermally exposed to the nerve agent VX. In the present study, an in vitro flow-through diffusion cell was utilized to evaluate the efficacy of RSDL for decontamination of VX exposed to human epidermis. In particular, the impact of timing in the initiation of decontamination and agent dilution in water was studied. The impact of the lipophilic properties of VX in the RSDL decontamination was additionally addressed by comparing chemical degradation in RSDL and decontamination efficacy between the VX and the hydrophilic OPC triethyl phosphonoacetate (TEPA). The epidermal membrane was exposed to 20, 75 or 90% OPC diluted in deionized water and the decontamination was initiated 5, 10, 30, 60 or 120min post-exposure. Early decontamination of VX with RSDL, initiated 5-10min after skin exposure, was very effective. Delayed decontamination initiated 30-60min post-exposure was less effective but still the amount of penetrated agent was significantly reduced, while further delayed start of decontamination to 120min resulted in very low efficacy. Comparing RSDL decontamination of VX with that of TEPA showed that the decontamination efficacy at high agent concentrations was higher for VX. The degradation mechanism of VX and TEPA during decontamination was dissected by 31P NMR spectroscopy of the OPCs following reactions with RSDL and its three nucleophile components. The degradation rate was clearly associated with the high pH of the specific solution investigated; i.e. increased pH resulted in a more rapid degradation. In addition, the solubility of the OPC in RSDL also influenced the degradation rate since the degradation of VX was significantly faster when the NMR analysis was performed in the organic solvent acetonitrile compared to water. In conclusion, we have applied the in vitro flow-through diffusion cell for evaluation of skin decontamination procedures of human epidermis exposed to OPCs. It was demonstrated that early decontamination is crucial for efficient mitigation of epidermal penetration of VX and that almost complete removal of the nerve agent from the skin surface is possible. Our data also indicate that the pH of RSDL together with the solubility of OPC in RSDL are of primary importance for the decontamination efficacy.