Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN), HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.

Inhibition of peroxynitrite- and peroxidase-mediated protein tyrosine nitration by imidazole-based thiourea and selenourea derivatives

In the present study, the synthesis and characterization of a series of N-methylimidazole-based thiourea and selenourea derivatives are described. The new compounds were also studied for their ability to inhibit peroxynitrite (PN)- and peroxidase-mediated nitration of protein tyrosine residues. It has been observed that the selenourea derivatives are more efficient than the thiourea-based compounds in the inhibition of protein nitration. The higher activity of selenoureas as compared to that of the corresponding thioureas can be ascribed to the zwitterionic nature of the selenourea moiety. Single crystal X-ray diffraction studies on some of the thiourea and selenourea derivatives reveal that the C S bonds in thioureas possess more of double bond character than the C=Se bonds in the corresponding selenoureas. Therefore, the selenium compounds can react with PN or hydrogen peroxide much faster than their sulfur analogues. The reactions of thiourea and selenourea derivatives with PN or hydrogen peroxide produce the corresponding sulfinic or seleninic acid derivatives, which upon elimination of sulfurous/selenous acids produce the corresponding N-methylimdazole derivatives.

Inhibition of type IA topoisomerase by a monoclonal antibody through perturbation of DNA cleavage-religation equilibrium

Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavagereligation equilibrium, increasing single-strand nicks and proteinDNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.

Inhibition of Mycobacterium tuberculosis RNA Polymerase by Binding of a Gre Factor Homo log to the Secondary Channel

Because of its essential nature, each step of transcription, viz., initiation, elongation, and termination, is subjected to elaborate regulation. A number of transcription factors modulate the rates of transcription at these different steps, and several inhibitors shut down the process. Many modulators, including small molecules and proteinaceous inhibitors, bind the RNA polymerase (RNAP) secondary channel to control transcription. We describe here the first small protein inhibitor of transcription in Mycobacterium tuberculosis. Rv3788 is a homolog of the Gre factors that binds near the secondary channel of RNAP to inhibit transcription. The factor also affected the action of guanosine pentaphosphate (pppGpp) on transcription and abrogated Gre action, indicating its function in the modulation of the catalytic center of RNAP. Although it has a Gre factor-like domain organization with the conserved acidic residues in the N terminus and retains interaction with RNAP, the factor did not show any transcript cleavage stimulatory activity. Unlike Rv3788, another Gre homolog from Mycobacterium smegmatis, MSMEG_6292 did not exhibit transcription-inhibitory activities, hinting at the importance of the former in influencing the lifestyle of M. tuberculosis.

Dimeric 1,3-Phenylene-bis(piperazinyl benzimidazole)s: Synthesis and Structure-Activity Investigations on their Binding with Human Telomeric G-Quadruplex DNA and Telomerase Inhibition Properties

Ligand-induced stabilization of G-quadruplex structures formed by the human telomeric DNA is an active area of research. The compounds which stabilize the G-quadruplexes often lead to telomerase inhibition. Herein we present the results of interaction of new monomeric and dimeric ligands having 1,3-phenylene-bis(piperazinyl benzimidazole) unit with G-quadruplex DNA (G4DNA) formed by human telomeric repeat d(G(3)T(2)A)(3)G(3)]. These ligands efficiently stabilize the preformed G4DNA in the presence of 100 mM monovalent alkali metal ions. Also, the G4DNA formed in the presence of low concentrations of ligands in 100 mM K+ adopts a highly stable parallel-stranded conformation. The G-quadruplexes formed in the presence of the dimeric compound are more stable than that induced by the corresponding monomeric counterpart. The dimeric ligands having oligo-oxyethylene spacers provide much higher stability to the preformed G4DNA and also exert significantly higher telomerase inhibition activity. Computational aspects have also been discussed.

Mutual inhibition and capacity sharing during parallel preparation of serial eye movements

Many common activities, like reading, scanning scenes, or searching for an inconspicuous item in a cluttered environment, entail serial movements of the eyes that shift the gaze from one object to another. Previous studies have shown that the primate brain is capable of programming sequential saccadic eye movements in parallel. Given that the onset of saccades directed to a target are unpredictable in individual trials, what prevents a saccade during parallel programming from being executed in the direction of the second target before execution of another saccade in the direction of the first target remains unclear. Using a computational model, here we demonstrate that sequential saccades inhibit each other and share the brain's limited processing resources (capacity) so that the planning of a saccade in the direction of the first target always finishes first. In this framework, the latency of a saccade increases linearly with the fraction of capacity allocated to the other saccade in the sequence, and exponentially with the duration of capacity sharing. Our study establishes a link between the dual-task paradigm and the ramp-to-threshold model of response time to identify a physiologically viable mechanism that preserves the serial order of saccades without compromising the speed of performance.

Anticancer Activity of Hydrogen-Bond-Stabilized Half-Sandwich RuII Complexes with Heterocycles

Neutral half-sandwich organometallic ruthenium(II) complexes of the type (?6-cymene)RuCl2(L)] (H1H10), where L represents a heterocyclic ligand, have been synthesized and characterized spectroscopically. The structures of five complexes were also established by single-crystal X-ray diffraction confirming a piano-stool geometry with ?6 coordination of the arene ligand. Hydrogen bonding between the N?H group of the heterocycle and a chlorine atom attached to Ru stabilizes the metalligand interaction. Complexes coordinated to a mercaptobenzothiazole framework (H1) or mercaptobenzoxazole (H6) showed high cytotoxicity against several cancer cells but not against normal cells. In vitro studies have shown that the inhibition of cancer cell growth involves primarily G1-phase arrest as well as the generation of reactive oxygen species (ROS). The complexes are found to bind DNA in a non-intercalative fashion and cause unwinding of plasmid DNA in a cell-free medium. Surprisingly, the cytotoxic complexes H1 and H6 differ in their interaction with DNA, as observed by biophysical studies, they either cause a biphasic melting of the DNA or the inhibition of topoisomerase IIa activity, respectively. Substitution of the aromatic ring of the heterocycle or adding a second hydrogen-bond donor on the heterocycle reduces the cytotoxicity.

Characterization of DNA topoisomerase I from Mycobacterium tuberculosis: DNA cleavage and religation properties and inhibition of its activity

Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg2+ dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg2+ co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg2+ for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Inhibition of the Interaction Between NS3 Protease and HCV IRES With a Small Peptide: A Novel Therapeutic Strategy

Recently, we have demonstrated that the protease domain of NS3 alone can bind specifically to hepatitis C virus (HCV) internal ribosome entry site (IRES) near the initiator AUG, dislodges human La protein and inhibits translation in favor of viral RNA replication. Here, by using a computational approach, the contact points of the protease on the HCV IRES were putatively mapped. A 30-mer NS3 peptide was designed from the predicted RNA-binding region that retained RNA-binding ability and also inhibited IRES-mediated translation. This peptide was truncated to 15 mer and this also demonstrated ability to inhibit HCV RNA-directed translation as well as replication. More importantly, its activity was tested in an in vivo mouse model by encapsulating the peptide in Sendai virus virosomes followed by intravenous delivery. The study demonstrates for the first time that the HCV NS3-IRES RNA interaction can be selectively inhibited using a small peptide and reports a strategy to deliver the peptide into the liver.

Non-neutral evolution in non-LEE-encoded type III effectors of attaching and effacing Escherichia coli

Attaching and effacing Escherichia coli (AEEC) employ type III secretion system (T3SS) to secrete effector proteins into host cells and regulate their function. Here we have investigated T3SS genes of AEEC for non-neutral evolution. Our analysis revealed non-neutral evolution in three genes (nleE1, nleB2 and nleD) which encode effector proteins. These genes are located outside the locus of enterocyte effacement (LEE). In general, non-LEE effector genes show greater deviation from neutral evolution than LEE effector genes. These results suggest that effector genes located outside LEE are under greater selection pressure than those present in LEE. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

In Vitro and in Vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes

Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo II alpha inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.

Charge-transfer-driven inclusion of neutral TCNQ molecules in the galleries of a layered double hydroxide: an experimental and computational study

The layered double hydroxides (LDH) or anionic clays are an important class of ion-exchange materials. They consist of positively charged brucite-like inorganic sheets with charge-compensating exchangeable anions in the interlamellar space. Here we show how neutral TCNQ (7,7,8,8-tetracyanoquinodimethane) molecules can be included within the galleries of an LDH. To do so, we exploit the fact that TCNQ is a good electron acceptor that forms donor acceptor complexes with a variety of donors. The electron donor aniline was intercalated into a Mg-Al LDH as p-aminobenzoate (AB) ions by a conventional ion-exchange reaction. We show here that neutral TCNQ molecules may be driven into the galleries of the layered solid by charge-transfer complex formation with the intercalated p-aminobenzoate anions. We use diffraction and spectroscopic measurements in combination with molecular dynamics simulations and quantum chemical calculations to establish the nature of interactions and arrangement of the charge-transfer complex within the galleries of the layered double hydroxide. Electrostatic interactions between the TCNQ molecules and the anchored AB ions, subsequent to charge transfer, are the driving force for the inclusion of TCNQ molecules in the galleries of the LDH.