Structure-Based Design of DevR Inhibitor Active against Nonreplicating Mycobacterium tuberculosis

Antitubercular treatment is directed against actively replicating organisms. There is an urgent need to develop drugs targeting persistent subpopulations of Mycobacterium tuberculosis. The DevR response regulator is believed to play a key role in bacterial dormancy adaptation during hypoxia. We developed a homology-based model of DevR and used it for the rational design of inhibitors. A phenylcoumarin derivative (compound 10) identified by in silico pharmacophore-based screening of 2.5 million compounds employing protocols with some novel features including a water-based pharmacophore query, was characterized further. Compound 10 inhibited DevR binding to target DNA, down-regulated dormancy genes transcription, and drastically reduced survival of hypoxic but not nutrient-starved dormant bacteria or actively growing organ ` isms. Our findings suggest that compound 10 ``locks'' DevR in an inactive conformation that is unable to bind cognate DNA and induce the dormancy regulon. These results provide proof-of-concept for DevR as a novel target to develop molecules with sterilizing activity against tubercle bacilli.

Mitochondria-Targeting Oxidovanadium(IV) Complex as a Near-IR Light Photocytotoxic Agent

Oxidovanadium(IV) complexes VO(L-1)(phen)]Cl (1) and VO(L-2)(L-3)]Cl (2), in which HL1 is 2-{(benzimidazol-2-yl)methylimino]-methyl}phenol (sal-ambmz), HL2 is 2-({1-(anthracen-9-yl)methyl]-benzimidazol-2-yl}methylimino)-met hyl]phenol (sal-an-ambmz), phen is 1,10-phenanthroline and L-3 is dipyrido3,2-a:2,3-c]phenazine (dppz) conjugated to a Gly-Gly-OMe dipeptide moiety, were prepared, characterized, and their DNA binding, photoinduced DNA-cleavage, and photocytotoxic properties were studied. Fluorescence microscopy studies were performed by using complex 2 in HeLa and HaCaT cells. Complex 1, structurally characterized by X-ray crystallography, has a vanadyl group in VO2N4 core with the VO2+ moiety bonded to N,N-donor phen and a N,N,O-donor Schiff base. Complex 2, having an anthracenyl fluorophore, showed fluorescence emission bands at 397, 419, and 443nm. The complexes are redox-active exhibiting the V(IV)/V(III) redox couple near -0.85V versus SCE in DMF 0.1M tetrabutylammonium perchlorate (TBAP). Complex 2, having a dipeptide moiety, showed specific binding towards poly(dAdT)(2) sequence. The dppz-Gly-Gly-OMe complex showed significant DNA photocleavage activity in red light of 705nm through a hydroxyl radical ((OH)-O-.) pathway. Complex 2 showed photocytotoxicity in HaCaT and HeLa cells in visible light (400-700nm) and red light (620-700nm), however, the complex was less toxic in the dark. Fluorescence microscopy revealed the localization of complex 2 primarily in mitochondria. Apoptosis was found to occur inside mitochondria (intrinsic pathway) caused by ROS generation.

Carbohydrate-Appended Tumor Targeting Iron(III) Complexes Showing Photocytotoxicity in Red Light

Glucose-appended photocytotoxic iron(III) complexes of a tridentate Schiff base phenolate ligand Fe(bpyag) (L)] (NO3) (1-3), where bpyag is N,N-bis(2- pyridylmethyl)-2-aminoethyl-beta-D-glucopyranoside and H2L is 3-(2-hydroxyphenylimino)-1-phenylbutan-1-one (H(2)phap) in 1, 3-(2-hydroxyphenylimino)-9-anthrylbutan-1-one (H(2)anap) 2, and 3- (2-hydroxyphenylimino)-1-pyrenylbutan-1-one (H(2)pyap) in 3, were synthesized and characterized. The complex Fe(dpma)(anapn(NO3) (4), having bis-(2-pyridylmethyl)benzylamine (dpma), in which the glucose moiety of bpyag is substituted by a phenyl group, was used as a control, and the complex Fe(dpma)(anap)](PF6) (4a) was structurally characterized by X-ray crystallography. The structure shows a FeN4O2 core in a distorted octahedral geometry. The high-spin iron(III) complexes with magnetic moment value of similar to 5.9 mu(B) showed a low-energy phenolate-to-Fe(III) charge-transfer (CT) absorption band as a shoulder near 500 nm with a tail extending to 700 nm and an irreversible Fe(III)-Fe(II) redox couple near -0.6 V versus saturated calomel electrode. The complexes are avid binders to calf thymus DNA and showed photocleavage of supercoiled pUC19 DNA in red (647 nm) and green (532 nm) light. Complexes 2 and 3 displayed significant photocytotoxicity in red light, with an IC50 value of similar to 20 mu M in HeLa and HaCaT cells, and no significant toxicity in dark. The cell death is via an apoptotic pathway, by generation of reactive oxygen species. Preferential internalization of the carbohydrate-appended complexes 2 and 3 was evidenced in HeLa cells as compared to the control complex 4. A 5-fold increase in the cellular uptake was observed for the active complexes in HeLa cells. The photophysical properties of the complexes are rationalized from the density functional theory calculations.

Homeostasis of functional maps in active dendrites emerges in the absence of individual channelostasis

The maintenance of ion channel homeostasis, or channelostasis, is a complex puzzle in neurons with extensive dendritic arborization, encompassing a combinatorial diversity of proteins that encode these channels and their auxiliary subunits, their localization profiles, and associated signaling machinery. Despite this, neurons exhibit amazingly stereotypic, topographically continuous maps of several functional properties along their active dendritic arbor. Here, we asked whether the membrane composition of neurons, at the level of individual ion channels, is constrained by this structural requirement of sustaining several functional maps along the same topograph. We performed global sensitivity analysis on morphologically realistic conductance-based models of hippocampal pyramidal neurons that coexpressed six well-characterized functional maps along their trunk. We generated randomized models by varying 32 underlying parameters and constrained these models with quantitative experimental measurements from the soma and dendrites of hippocampal pyramidal neurons. Analyzing valid models that satisfied experimental constraints on all six functional maps, we found topographically analogous functional maps to emerge from disparate model parameters with weak pairwise correlations between parameters. Finally, we derived a methodology to assess the contribution of individual channel conductances to the various functional measurements, using virtual knockout simulations on the valid model population. We found that the virtual knockout of individual channels resulted in variable, measurement and location-specific impacts across the population. Our results suggest collective channelostasis as a mechanism behind the robust emergence of analogous functional maps and have significant ramifications for the localization and targeting of ion channels and enzymes that regulate neural coding and homeostasis.

Metal Complexes of Curcumin for Cellular Imaging, Targeting, and Photoinduced Anticancer Activity

CONSPECTUS: Curcumin is a polyphenolic species. As an active ingredient of turmeric, it is well-known for its traditional medicinal properties. The therapeutic values include antioxidant, anti-inflammatory, antiseptic, and anticancer activity with the last being primarily due to inhibition of the transcription factor NF-kappa B besides affecting several biological pathways to arrest tumor growth and its progression. Curcumin with all these positive qualities has only remained a potential candidate for cancer treatment over the years without seeing any proper usage because of its hydrolytic instability involving the diketo moiety in a cellular medium and its poor bioavailability. The situation has changed considerably in recent years with the observation that curcumin in monoanionic form could be stabilized on binding to a metal ion. The reports from our group and other groups have shown that curcumin in the metal-bound form retains its therapeutic potential. This has opened up new avenues to develop curcumin-based metal complexes as anticancer agents. Zinc(II) complexes of curcumin are shown to be stable in a cellular medium. They display moderate cytotoxicity against prostate cancer and neuroblastoma cell lines. A similar stabilization and cytotoxic effect is reported for (arene)ruthenium(II) complexes of curcumin against a variety of cell lines. The half-sandwich 1,3,5-triaza-7-phosphatricyclo-3.3.1.1]decane (RAPTA)-type ruthenium(II) complexes of curcumin are shown to be promising cytotoxic agents with low micromolar concentrations for a series of cancer cell lines. In a different approach, cobalt(III) complexes of curcumin are used for its cellular delivery in hypoxic tumor cells using intracellular agents that reduce the metal and release curcumin as a cytotoxin. Utilizing the photophysical and photochemical properties of the curcumin dye, we have designed and synthesized photoactive curcumin metal complexes that are used for cellular imaging by fluorescence microscopy and damaging the cancer cells on photoactivation in visible light while being minimally toxic in darkness. In this Account, we have made an attempt to review the current status of the chemistry of metal curcumin complexes and present results from our recent studies on curcumin complexes showing remarkable in vitro photocytotoxicity. The undesirable dark toxicity of the complexes can be reduced with suitable choice of the metal and the ancillary ligands in a ternary structure. The complexes can be directed to specific subcellular organelles. Selectivity by targeting cancer cells over normal cells can be achieved with suitable ligand design. We expect that this methodology is likely to provide an impetus toward developing curcumin-based photochemotherapeutics for anticancer treatment and cure.

The role of lysine-41 in RNase A catalysis - A quantum chemical study on the active site ligand complex

Several mechanisms have been proposed to explain the action of enzymes at the atomic level. Among them, the recent proposals involving short hydrogen bonds as a step in catalysis by Gerlt and Gassman [1] and proton transfer through low barrier hydrogen bonds (LBHBs) [2, 3] have attracted attention. There are several limitations to experimentally testing such hypotheses, Recent developments in computational methods facilitate the study of active site-ligand complexes to high levels of accuracy, Our previous studies, which involved the docking of the dinucleotide substrate UpA to the active site of RNase A [4, 5], enabled us to obtain a realistic model of the ligand-bound active site of RNase A. From these studies, based on empirical potential functions, we were able to obtain the molecular dynamics averaged coordinates of RNase A, bound to the ligand UpA. A quantum mechanical study is required to investigate the catalytic process which involves the cleavage and formation of covalent bonds. In the present study, we have investigated the strengths of some of the hydrogen bonds between the active site residues of RNase A and UpA at the ab initio quantum chemical level using the molecular dynamics averaged coordinates as the starting point. The 49 atom system and other model systems were optimized at the 3-21G level and the energies of the optimized systems were obtained at the 6-31G* level. The results clearly indicate the strengthening of hydrogen bonds between neutral residues due to the presence of charged species at appropriate positions. Such a strengthening manifests itself in the form of short hydrogen bonds and a low barrier for proton transfer. In the present study, the proton transfer between the 2'-OH of ribose (from the substrate) and the imidazole group from the H12 of RNase A is influenced by K41, which plays a crucial role in strengthening the neutral hydrogen bond, reducing the barrier for proton transfer.

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalentlv associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hGG alpha and hCG beta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Go-expression of both subunits in the same cell resulted in secretion of heterodimeric hGG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hGG. The le level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermenter. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hGG and is suitable for structure-function studies of the hormone.

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.

Transient pulse evolution of active mode locking in an intracavity frequency-doubled laser

The theory of transient mode locking for an active modulator in an intracavity frequency-doubled laser is presented. The theory is applied to mode-locked and intracavity frequency-doubled Nd:YAG laser and the mode-locked pulse width is plotted as a function of number of round trips inside the cavity. It is found that the pulse compression is faster and the system takes a very short time to approach the steady state in the presence of a second harmonic generating crystal inside the laser cavity. The effect of modulation depth and the second harmonic conversion efficiency on the temporal behavior of the pulse width is discussed. Journal of Applied Physics is copyrighted by The American Institute of Physics.

Sensible heat fluxes during the active and break phases of the southwest monsoon over the Indian region

Based on the theory given by Saltzman and Ashe (1976), sensible heat fluxes are calculated for the active and break phases of the southwest monsoon over the Indian region. The conclusion drawn is that the sensible heat flux is generally larger during the break monsoon situation when compared with that for the active monsoon situation. The synoptic heat flux is negligible when compared with mean and diurnal heat fluxes over the Indian region even during the monsoon season.

Molecular structure of Boc-Aib-Aib-Phe-Met-NH2·DMSO. A fragment of a biologically active enkephalin analogue

The tetrapeptide t-butyloxycarbonyl--aminoisobutyryl--aminoisobutyryl-L- phenylalanyl-L-methionyl amide crystallizes in the orthorhombic space group P212121 with a= 9.096, b= 18.067, c= 21.701 Å and Z= 4. The crystals contain one molecule of dimethyl sulphoxide (DMSO) associated with each peptide. The structure has been solved by direct methods and refined to an R value of 0.103 for 2 672 observed reflections. The peptide adopts a distorted 310 helical structure stabilized by two intramolecular 4 1 hydrogen bonds between the Boc CO and Aib(1) CO groups and the NH groups of Phe(3) and Met(4), respectively. A long hydrogen bond (N O = 3.35 Å) is also observed between Aib(2) CO and one of the terminal amide hydrogens. The DMSO molecule is strongly hydrogen bonded to the Aib(1) NH group. The solid-state conformation agrees well with proposals made on the basis of n.m.r. studies in solution.

Biochemical and structural characterization of residue 96 mutants of Plasmodium falciparum triosephosphate isomerase: active-site loop conformation, hydration and identification of a dimer-interface ligand-binding site

Plasmodium falciparum TIM (PfTIM) is unique in possessing a Phe residue at position 96 in place of the conserved Ser that is found in TIMs from the majority of other organisms. In order to probe the role of residue 96, three PfTIM mutants, F96S, F96H and F96W, have been biochemically and structurally characterized. The three mutants exhibited reduced catalytic efficiency and a decrease in substrate-binding affinity, with the most pronounced effects being observed for F96S and F96H. The k(cat) values and K-m values are (2.54 +/- 0.19) x 10(5) min(-1) and 0.39 +/- 0.049 mM, respectively, for the wild type; (3.72 +/- 0.28) x 10(3) min(-1) and 2.18 +/- 0.028 mM, respectively, for the F96S mutant;(1.11 +/- 0.03) x 10(4) min(-1) and 2.62 +/- 0.042 mM, respectively, for the F96H mutant; and (1.48 +/- 0.05) x 10(5) min(-1) and 1.20 +/- 0.056 mM, respectively, for the F96W mutant. Unliganded and 3-phosphoglycerate (3PG) complexed structures are reported for the wild-type enzyme and the mutants. The ligand binds to the active sites of the wild-type enzyme (wtPfTIM) and the F96W mutant, with a loop-open state in the former and both open and closed states in the latter. In contrast, no density for the ligand could be detected at the active sites of the F96S and F96H mutants under identical conditions. The decrease in ligand affinity could be a consequence of differences in the water network connecting residue 96 to Ser73 in the vicinity of the active site. Soaking of crystals of wtPfTIM and the F96S and F96H mutants resulted in the binding of 3PG at a dimer-interface site. In addition, loop closure at the liganded active site was observed for wtPfTIM. The dimer-interface site in PfTIM shows strong electrostatic anchoring of the phosphate group involving the Arg98 and Lys112 residues of PfTIM.

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.

Chemical probes into the active centre of a heme thiolate monoxygenase

Linalool-8-monoxygenase, a typical bacterial P-450 heme thiolase, shows a high degree of substrate specificity towards linalool. The active site of the pure enzyme has been probed with a large number of substrate analogues with systematic alterations or conformational variations in the linalool molecule. The comparison of three parameters, the mo→mos conversion of the enzyme as a result of substrate binding monitored at 392 nm, theK D of the analogues giving information about energies of association and the relative turnover as substrate have given information about the space-filling characteristics of the substrates in the enzyme cleft, the number of contacts the molecules make with the respective domains of the enzyme and the distance of the site undergoing hydroxylation from the oxygen site, respectively. The data permit the conclusion that linalool makes contact with the enzyme by hydrogen bonding with the hydroxyl group as well through hydrophobic association with all the eight carbons carrying hydrogen in the molecules.

Transient pulse evolution of active-mode locking in an intra-cavity frequency-doubled laser

The theory of transient mode locking for an active modulator in an intracavity frequency-doubled laser is presented. The theory is applied to mode-locked and intracavity frequency-doubled Nd:YAG laser and the mode-locked pulse width is plotted as a function of number of round trips inside the cavity. It is found that the pulse compression is faster and the system takes a very short time to approach the steady state in the presence of a second harmonic generating crystal inside the laser cavity. The effect of modulation depth and the second harmonic conversion efficiency on the temporal behavior of the pulse width is discussed.