Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

Revisiting the regiospecificity of Burkholderia xenovorans LB400 biphenyl dioxygenase toward 2,2'-dichlorobiphenyl and 2,3,2',3'-tetrachlorobiphenyl

2,2'-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites (1 and 2). The most abundant metabolite, 1, was previously identified as 2,3-dihydroxy-2'-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2',3'-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2'-CB and 2,3,2',3'-CB, thus providing new insights on the mechanism by which 2,2'-CB is dehalogenated to generate 2,3-dihydroxy-2'-chlorobiphenyl. We reacted 2,2'-CB with the BPDO variant p4, which produces a larger proportion of metabolite 2. The structure of this compound was determined as cis-3,4-dihydro-3,4-dihydroxy-2,2'-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2'-CB-d(8) was determined to be a dihydroxychlorobiphenyl-d(7) by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2',3'-CB by LB400 BPDO was identified by NMR as cis-4,5-dihydro-4,5-dihydroxy-2,3,2',3'-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2'-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2'-CB and 2,3,2,',3'-CB disfavors the dioxygenation of the chlorine-free ortho-meta carbons 5 and 6 for both congeners.     

Proteolytic processing and translation initiation: two independent mechanisms for the expression of the Sendai virus Y proteins

The four Sendai virus C-proteins (C', C, Y1, and Y2) represent an N-terminal nested set of non-structural proteins whose expression modulates both the readout of the viral genome and the host cell response. In particular, they modulate the innate immune response by perturbing the signaling of type 1 interferons. The initiation codons for the four C-proteins have been mapped in vitro, and it has been proposed that the Y proteins are initiated by ribosomal shunting. A number of mutations were reported that significantly enhanced Y expression, and this was attributed to increased shunt-mediated initiation. However, we demonstrate that this arises due to enhanced proteolytic processing of C', an event that requires its very N terminus. Curiously, although Y expression in vitro is mediated almost exclusively by initiation, Y proteins in vivo can arise both by translation initiation and processing of the C' protein. To our knowledge this is the first example of two apparently independent pathways leading to the expression of the same polypeptide chain. This dual pathway explains several features of Y expression.     

Lassa virus infection of human dendritic cells and macrophages is productive but fails to activate cells

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), an old-world Arenavirus. Little is known about the immune responses that occur during the disease, but protection seems to be linked to the induction of cellular responses specific for viral glycoproteins. Conversely, severe Lassa fever may be associated with immunosuppression. We studied the infection of human dendritic cells (DC) and macrophages (MP) by LV. Both these cell types are susceptible to LV infection. Viral nucleoprotein was detected in DC and MP, and high and moderate viral titers were obtained with culture supernatants of DC and MP, respectively. LV did not induce apoptosis in DC and MP. These cells were not activated by LV infection. No change was observed in the expression of surface molecules involved in activation, costimulation, adhesion, and Ag presentation following LV infection, or in the functional properties of DC. Inflammatory cytokine production was not detected at the mRNA or protein level after LV infection of DC and MP. Thus, MP, and particularly DC, are crucial targets for LV and are probably involved in the early replication of LV from the initial site of infection. The lack of activation and maturation of cells following infection may be associated with the immunosuppression observed in severe LV infection.     

Nucleotide binding to DNA gyrase causes loss of DNA wrap

DNA gyrase negatively supercoils DNA in a reaction coupled to the binding and hydrolysis of ATP. Limited supercoiling can be achieved in the presence of the non-hydrolysable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (ADPNP). In order to negatively supercoil DNA, gyrase must wrap a length of DNA around itself in a positive sense. In previous work, the effect of ADPNP on the gyrase-DNA interaction has been assessed but has produced conflicting results; the aim of this work was to resolve this conflict. We have probed the wrapping of DNA around gyrase in the presence and in the absence of ADPNP using direct observation by atomic force microscopy (AFM). We confirm that gyrase indeed generates a significant curvature in DNA in the absence of nucleotide and we show that the addition of ADPNP leads to a complete loss of wrap. These results have been corroborated using a DNA relaxation assay involving topoisomerase I. We have re-analysed previous hydroxyl-radical footprinting and crystallography data, and highlight the fact that the gyrase-DNA complex is surprisingly asymmetric in the absence of nucleotide but is symmetric in the presence of ADPNP. We suggest a revised model for the conformation of DNA bound to the enzyme that is fully consistent with these AFM data, in which a closed loop of DNA is stabilised by the enzyme in the absence of ADPNP and is lost in the presence of nucleotide.     

Cellular and biochemical features of skeletal muscle in obese Yucatan minipigs

Objective: To examine cellular and biochemical features of skeletal muscle in response to dietary‐induced obesity in a novel Yucatan minipig model of childhood obesity. Research Methods and Procedures: From 4 to 16 months of age, minipigs were fed either a recommended human‐type diet (NF; n = 4) or were overfed a western‐type diet with saturated fat and high‐glycemic index carbohydrates (OF, n = 4). Muscle samples (biceps femoris) were histochemically stained for the identification of intramuscular adipocytes, intramyocellular lipid aggregates (oil red O), and myofiber types (myosin ATPase, succinate dehydrogenase). Gene expressions and/or activities of factors involved in lipogenesis, lipolysis, or energetic metabolism were quantified in muscle. Results: Cross‐sectional areas of myofibers paralleled pig body weight (r = 0.86, p < 0.01). The size of intramuscular adipocytes, the relative proportion of oil red O‐stained fibers, and total muscle lipid content tended (p ≤ 0.10) to increase in response to OF diet. Hormone‐sensitive lipase, carnitine palmityl transferase‐I, and uncoupling protein 2 mRNA levels were lower (p < 0.05) in OF pigs than in NF pigs. Activities of β‐hydroxyacyl‐coenzyme A dehydrogenase and citrate synthase assessing post‐carnitine palmityl transferase I events and the proportion of oxidative myofibers were not altered by OF diet. Activity and gene expression of fatty acid synthase were lower (p < 0.02) in OF pigs than in NF pigs. Discussion: Overfeeding in Yucatan minipigs reduced the expression levels of three catabolic steps in skeletal muscle that are involved also in the etiology of human obesity.     

Antiviral activity of benzimidazole derivatives. II. Antiviral activity of 2-phenylbenzimidazole derivatives

Seventy-six 2-phenylbenzimidazole derivatives were synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. The most commonly affected viruses were, in decreasing order, CVB-2, BVDV, Sb-1, HSV-1, and YFV, while HIV-1 and VSV were not affected, and RSV, VV and Reo-1 were only susceptible to a few compounds. Thirty-nine compounds exhibited high activity (EC₅₀ = 0.1–10 μM) against at least one virus, and four of them were outstanding for their high and selective activity against VV (24, EC₅₀ = 0.1 μM) and BVDV (50, 51, and 53 with EC₅₀ = 1.5, 0.8, and 1.0 μM, respectively). The last compounds inhibited at low micromolar concentrations the NS5B RdRp of BVDV and also of HCV, the latter sharing structural similarity with the former. The considered compounds represent attractive leads for the development of antiviral agents against poxviruses, pestiviruses and even HCV, which are important human and veterinary pathogens.     

Potassium Channel Silencing by Constitutive Endocytosis and Intracellular Sequestration

Tandem of P domains in a weak inwardly rectifying K⁺ channel 1 (TWIK1) is a K⁺ channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT₁R or adrenoreceptor α2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G_i protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.