Crystal structure of a putative aspartic proteinase domain of the Mycobacterium tuberculosis cell surface antigen PE_PGRS16

We report the crystal structure of the first prokaryotic aspartic proteinase-like domain identified in the genome of Mycobacterium tuberculosis. A search in the genomes of Mycobacterium species showed that the C-terminal domains of some of the PE family proteins contain two classic DT/SG motifs of aspartic proteinases with a low overall sequence similarity to HIV proteinase. The three-dimensional structure of one of them, Rv0977 (PE_PGRS16) of M. tuberculosis revealed the characteristic pepsinf-old and catalytic site architecture. However, the active site was completely blocked by the N-terminal His-tag. Surprisingly, the enzyme was found to be inactive even after the removal of the N-terminal His-tag. A comparison of the structure with pepsins showed significant differences in the critical substrate binding residues and in the flap tyrosine conformation that could contribute to the lack of proteolytic activity of Rv0977. (C) 2013 The Authors. Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies. All rights reserved.

DNA binders in clinical trials and chemotherapy

Cancer has always been a dreadful disease and continues to attract extensive research investigations. Various targets have been identified to restrain cancer. Among these DNA happens to be the most explored one. A wide variety of small molecules, often referred to as `ligands', has been synthesized to target numerous structural features of DNA. The sole purpose of such molecular design has been to interfere with the transcriptional machinery in order to drive the cancer cell toward apoptosis. The mode of action of the DNA targeting ligands focuses either on the sequence-specificity by groove binding and strand cleavage, or by identifying the morphologically distinct higher order structures like that of the G-quadruplex DNA. However, in spite of the extensive research, only a tiny fraction of the molecules have been able to reach clinical trials and only a handful are used in chemotherapy. This review attempts to record the journey of the DNA binding small molecules from its inception to cancer therapy via various modifications at the molecular level. Nevertheless, factors like limited bioavailability, severe toxicities, unfavorable pharmacokinetics etc. still prove to be the major impediments in the field which warrant considerable scope for further research investigations. (C) 2014 Published by Elsevier Ltd.

Oxovanadium(IV) complexes of curcumin for cellular imaging and mitochondria targeted photocytotoxicity

Oxovanadium(IV) complexes VO(R-tpy)(cur)](ClO4) (1, 2) of curcumin (Hcur) and terpyridine ligands (R-tpy) where R is phenyl (phtpy in 1) or p-triphenylphosphonium methylphenyl bromide (C6H4CH2PPh3Br) (TPP-phtpy in 2) were prepared and characterized and their DNA photocleavage activity, photocytotoxicity and cellular localization in cancer cells (HeLa and MCF-7) were studied. Acetylacetonate (acac) complexes VO(R-tpy)(acac)](ClO4) of phtpy (3) and TPP-phtpy (4) were prepared and used as the control species. These complexes showed efficient cleavage of pUC19 DNA in visible light of 454 nm and near-IR light of 705 rim. Complexes 1 and 2 showed significant photocytotoxicity in visible light of 400-700 nm. FACS analysis showed sub-G1/G0 phase cell-cycle arrest in cancer cells when treated with 1 and 2 in visible light in comparison with the dark controls. Fluorescence microscopic studies revealed specific localization of the p-triphenylphosphonium complex 2 in the mitochondria of MCF-7 cancer cells whereas no such specificity was observed for complex 1.

Synthesis, characterization and reactivity studies of electrophilic ruthenium(II) complexes: a study of H-2 activation and labilization

Reaction of 2,2'-bipyridine (bpy) with dinuclear complexesRuCl(dfppe)(mu-Cl)(3)Ru(dmso-S)(3)](dfppe = 1,2-bis(dipentafluorophenyl phosphino)ethane (C6F5)(2)PCH2CH2P(C6F5)(2); dmso = dimethyl sulfoxide) (1) or RuCl(dfppe)(mu-Cl)(3)RuCl(dfppe)] (2) affords the mononuclear species trans-RuCl2(bpy)(dfppe)] (3). Using this precursor complex (3), a series of new cationic Ru(II) electrophilic complexes RuCl(L)(bpy)(dfppe)]Z] (L = P(OMe)(3) (5), PMe3 (6), CH3CN (7), CO (8), H2O (9); Z = OTf (5, 6, 7, 8), BAr4F (9) have been synthesized via abstraction of chloride by AgOTf or NaBAr4F in the presence of L. Complexes 5 and 6 were converted into the corresponding isomeric hydride derivatives RuH(PMe3)(bpy)(dfppe)]OTf] (10a, 10b) and RuH(P(OMe)(3))(bpy)(dfppe)]OTf] (11a, 11b) respectively, when treated with NaBH4. Protonation of the cationic monohydride complex (11a) with HOTf at low temperatures resulted in H-2 evolution accompanied by the formation of either solvent or triflate bound six coordinated species Ru(S)(P(OMe)(3))(bpy)(dfppe)]OTf](n) (S = solvent (n = 2), triflate (n = 1)] (13a/13b); these species have not been isolated and could not be established with certainty. They (13a/13b) were not isolated, instead the six-coordinated isomeric aqua complexes cis-(Ru(bpy)(dfppe)(OH2)(P(OMe)(3))]OTf](2) (14a/14b) were isolated. Reaction of the aqua complexes (14a/14b) with 1 atm of H-2 at room temperature in acetone-d(6) solvent resulted in heterolytic cleavage of the H-H bond. Results of the studies on H-2 lability and heterolytic activation using these complexes are discussed. The complexes 3, 5, 11a, and 14a have been structurally characterized.

Useful approach to the synthesis of aryl thio- and selenoglycosides in the presence of rongalite

A simple, mild, and cost effective methodology has been developed for the synthesis of aryl thio-and selenoglycosides from glycosyl halides and diaryl dichalcogenides. Diaryl dichalcogenides undergo reductive cleavage in the presence of rongalite (HOCH2SO2Na) to generate a chalcogenide anion in situ followed by reaction with glycosyl halides to furnish the corresponding aryl thio- and selenoglycosides in excellent yields. Using this protocol, synthesis of 4-methyl-7-thioumbelliferyl-beta-D-cellobioside (MUS-CB), a fluorescent non-hydrolyzable substrate analogue for cellulases has been achieved. (C) 2014 Elsevier Ltd. All rights reserved.

Oxovanadium(IV) catecholates of terpyridine bases for cellular imaging and photocytotoxicity in red light

Oxovanadium(IV) catecholates of terpyridyl bases, viz. VO(cat)(L)] (L - phtpy, 1; stpy, 2) and VO(dopa-NBD)(L)] (L = phtpy, 3; stpy, 4), where cat is benzene-1,2-diolate, dopa-NBD is 4-(2-(4-nitrobenzoc]1,2,5]oxadiazol-7-ylamino)ethyl)benzene-1,2-di olate, phtpy is (4'-phenyl)-2,2':6',2 `'-terpyridine and stpy is (2,2':6',2 `'-terpyridin-4'-oxy)ethyl-beta-D-glucopyranoside, were prepared and characterized, and their DNA binding, DNA photo-cleavage activity, photocytotoxicity in red light (600-720 nm), cellular uptake and intracellular localization behaviour were studied. The complexes showed an intense ligand-to-metal charge transfer (LMCT) band at similar to 500 nm. The sugar appended complexes 2 and 4 showed significant uptake into the cancer cells. The dopa-NBD complexes 3 and 4 showing green emission were used for cellular imaging. The complexes showed diffused cellular localization mainly in the cytosol and to a lesser extent into the nucleus as evidenced from the confocal microscopy study. Complexes 1-4 showed significant photocytotoxicity in the PDT spectral window giving low IC50 values, while remaining relatively non-toxic in dark.

Photoactivated cytotoxicity of ferrocenyl-terpyridine oxovanadium(IV) complexes of curcuminoids

Oxovanadium(IV) complexes, viz. VO(Fc-tpy)(Curc)](ClO4) (1), VO(Fc-tpy)(bDHC)](ClO4) (2), VO(Fc-tpy)(bDMC)](ClO4) (3) and VO(Ph-tpy)(Curc)](ClO4) (4), of 4'-ferrocenyl-2,2':6',2 `'-terpyridine (Fc-tpy) and 4'-phenyl-2,2':6',2 `'-terpyridine (Ph-tpy) and monoanionic curcumin (Curc), bis-dehydroxycurcmin (bDHC) and bis-demethoxycurcumin (bDMC) were prepared, characterized and their photo-induced DNA cleavage activity and photocytotoxicity in visible light studied. The ferrocenyl complexes 1-3 showed an intense metal-to-ligand charge transfer band near 585 nm in DMF and displayed Fc(+)/Fc and V(IV)/V(III) redox couples near 0.65 V and -1.05 V vs. SCE in DMF-0.1 M TBAP. The complexes as avid binders to calf thymus DNA showed significant photocleavage of plasmid DNA in red light of 647 nm forming (OH)-O-center dot radicals. The complexes showed photocytotoxicity in HeLa and Hep G2 cancer cells in visible light of 400-700 nm with low dark toxicity. ICP-MS and fluorescence microscopic studies exhibited significant cellular uptake of the complexes within 4 h of treatment with complexes. The treatment with complex 1 resulted in the formation of reactive oxygen species inside the HeLa cells which was evidenced from the DCFDA assay. (C) 2014 Elsevier Masson SAS. All rights reserved.

Metallacyclocumulenes: A Theoretical Perspective on the Structure, Bonding, and Reactivity

CONSPECTUS: Transition metals help to stabilize highly strained organic fragments. Metallacycles, especially unsaturated ones, provide much variety in this area. We had a sustained interest in understanding new C-C bond formation reactions affected by binuclear transition metal fragments Cp2M. One such study led to the exploration of the bimetallic C-C cleavage and coupled complexes, where the acetylide ligands bridge two metal atoms. The underlying M-C interaction in these complexes inspired the synthesis of a five-membered cyclocumulene complex, which opened a new phase in organometallic chemistry. The metallacyclocumulene produces a variety of C-C cleavage and coupled products including a radialene complex. Group 4 metallocenes have thus unlocked a fascinating chemistry by stabilizing strained unsaturated C4 organic fragments in the form of five-membered metallacyclocumulenes, metallacyclopentynes, and metallacycloallenes. Over the years, we have carried out a comprehensive theoretical study to understand the unusual stability and reactivity of these metallacycles. The unique (M-C-beta) interaction of the internal carbon atoms with the metal atom is the reason for unusual stability of the metallacycles. We have also shown that there is a definite dependence of the C-C coupling and cleavage reactions on the metal of metallacyclocumulenes. It demonstrates unexpected reaction pathways for these reactions. Based on this understanding, we have predicted and unraveled the stabilization factors of an unusual four-membered metallacycloallene complex. Indeed, our prediction about a four-membered heterometallacycle has led to an interesting bonding situation, which is experimentally realized. This type of M-C bonding is intriguing from a fundamental perspective and has great relevance in synthesizing unusual structures with interesting properties. In this Account, we first give a short prologue of what led to the present study and describe the salient features of the structure and bonding of the metallacyclocumulenes. The unusual reaction pathway of this metallacycle is explored next. Similar features of the metallacyclopentynes and metallacycloallenes are briefly mentioned. Then, we discuss the exploitation of the unique M-C bonding to design some exotic molecules such as a four-membered metallacycloallene complex. Our efforts to build a conceptual framework to understand these metallacycles and to exploit their chemistry continue.

The effect of gamma-gamma ` interface on the tensile and shear strengths of nickel-based superalloys: A first-principles study

First-principles density functional theory has been used to evaluate the shear and cleavage strength in terms of Griffith work and generalized stacking fault energy (GSF) of (001) plane for gamma, gamma' and gamma-gamma' system as a function of distance from the gamma/gamma' interface. Calculation of Griffith work suggests higher cleavage energy for bulk gamma as compared to gamma' while the GSF calculation suggests higher shear strength for bulk gamma' as compared to gamma. It has been found that the shear strength of the cubic plane of the gamma/gamma' interface is marginally lower than those of bulk gamma and gamma' phases. (C) 2014 Elsevier B.V. All rights reserved.

Structure-specific nuclease activity of RAGs is modulated by sequence, length and phase position of flanking double-stranded DNA

RAGs (recombination activating genes) are responsible for the generation of antigen receptor diversity through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. In addition to its physiological property, wherein RAG functions as a sequence-specific nuclease, it can also act as a structure-specific nuclease leading to genomic instability and cancer. In the present study, we investigate the factors that regulate RAG cleavage on non-B DNA structures. We find that RAG binding and cleavage on heteroduplex DNA is dependent on the length of the double-stranded flanking region. Besides, the immediate flanking double-stranded region regulates RAG activity in a sequence-dependent manner. Interestingly, the cleavage efficiency of RAGs at the heteroduplex region is influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights on the regulation of the pathological functions of RAGs.

Remarkable Selectivity and Photo-Cytotoxicity of an Oxidovanadium(IV) Complex of Curcumin in Visible Light

Oxidovanadium(IV) complexes of 2-(2'-pyridyl)-1,10-phenanthroline (pyphen), viz. VO(pyphen)(acac)](ClO4) (1), VO(pyphen)(anacac)](ClO4) (2) and VO(pyphen)(cur)](ClO4) (3), where acac is acetylacetonate (in 1), anacac is anthracenylacetylacetonate (in 2) and cur is curcumin monoanion (in 3) were synthesized, characterized and their photo-induced DNA cleavage activities and photo-cytotoxicities studied. The complexes are 1: 1 electrolytes in DMF. The one-electron paramagnetic complexes show a d-d band near 760 nm in DMF. Complexes 2 and 3 are blue and green emissive, respectively, in DMSO. The complexes exhibit irreversible V-IV/V-III reductive responses near -1.1 V and V-V/V-IV oxidative responses near 0.85 V vs. SCE in DMF-0.1 M TBAP. Complexes 2 and 3 display significant and selective photo-cytotoxicity upon irradiation with visible light giving an IC50 value of about 5 mu M against HeLa and MCF-7 cancer cells; they are significantly less-toxic against normal 3T3 control cells and in the absence of light. Complex 1 was used as a control. Both cytosolic and nuclear localization of the complexes were observed on the basis of fluorescence imaging. The complexes, avid binders to calf thymus (ct) DNA, were found to photocleave supercoiled pUC19 DNA upon irradiation with near-IR light (785 nm) by generating hydroxyl radical (OH) as the reactive oxygen species (ROS). Cell death events noted with HeLa and MCF-7 cell lines likely are attributable to apoptotic pathways involving light-assisted generation of intracellular ROS.

Individual and combined additions of calcium and antimony on microstructure and mechanical properties of squeeze-cast AZ91D magnesium alloy

The effects of combined additions of Ca and Sb on the microstructure and tensile properties of AZ91D alloy fabricated by squeeze-casting have been investigated. For comparison, the same has also been studied with and without individual additions of Ca and Sb. The results indicate that both individual and combined additions refine the grain size and beta-Mg17Al12 phase, which is more pronounced with combined additions. Besides alpha-Mg and beta-Mg17Al12 phases, a new reticular Al2Ca and rod-shaped Mg3Sb2 phases are formed following individual additions of Ca and Sb in the AZ91D alloy. With combined additions, an additional Ca2Sb phase is formed suppressing Mg3Sb2 phase. Additions of both Ca and Sb increase yield strength (YS) at both ambient and elevated temperatures up to 200 degrees C. However, both ductility and ultimate tensile strength (UTS) decrease first up to 150 degrees C and then increase at 200 degrees C. The increase in YS is attributed to the refinement of grain size, whereas, ductility and UTS are deteriorated by the presence of brittle Al2Ca, Mg3Sb2 and Ca2Sb phases. The best tensile properties are obtained in the AZXY9110 alloy owing to the presence of lesser amount of brittle Al2Ca and Ca2Sb phases resulted from the optimum content of 1.0Ca and 0.3Sb (wt%). The fracture surface of the tensile specimen tested at ambient temperature reveals cleavage failure that changes to quasi-cleavage at 200 degrees C. The squeeze-cast alloys exhibited better tensile properties as compared to that of the gravity-cast alloys nullifying the detrimental effects of Ca and/or Sb additions. (C) 2014 Elsevier B.V. All rights reserved.

A Kunitz-type serine protease inhibitor from Butea monosperma seed and its influence on developmental physiology of Helicoverpa armigera

A protease inhibitor from the seeds of Butea monosperma (BmPI) was purified, characterized and studied for its influence on developmental physiology of Helicover-pa armigera. BmPI on two-dimensional separations indicated the presence of a 14 kDa protein with an isoelectric point in the acidic region (pl 5.6). Multiple Sequence Analysis data suggested that the BmPI contains a sequence motif which is conserved in various trypsin and chymotrypsin inhibitors of Kunitz-type. The inhibitor exhibited trypsin inhibitory activity in a broad range of pH (4-10) and temperature (10-80 degrees C). The enzyme kinetic studies revealed BmPI as a competitive inhibitor with a K-i value of 1.2 x 10(-9) M. In vitro studies with BmPI indicated measurable inhibitory activity on total gut proteolytic enzymes of H. armigera (IC(50)2.0 mu g/ml) and bovine trypsin. BmPI supplemented artificial diet caused dose dependent mortality and reduction in growth and weight. The fertility and fecundity of H. armigera, declined whereas the larval-pupal duration of the insect life cycle extended. These detrimental effects on H. armigera suggest the usefulness of BmPl in insect pest management of food crops. (C) 2014 Elsevier Ltd. All rights reserved.

Remarkable enhancement in photocytotoxicity and hydrolytic stability of curcumin on binding to an oxovanadium(IV) moiety

Oxovanadium(IV) complexes of polypyridyl and curcumin-based ligands, viz. VO(cur)(L)Cl] (1, 2) and VO(scur)(L)Cl] (3, 4), where L is 1,10-phenanthroline (phen in 1 and 3), dipyrido3,2-a:2',3'-c]phenazine (dppz in 2 and 4), Hcur is curcumin and Hscur is diglucosylcurcumin, were synthesized and characterized and their cellular uptake, photocytotoxicity, intracellular localization, DNA binding, and DNA photo-cleavage activity studied. Complex VO(cur)(phen)Cl] (1) has (VN2O3Cl)-N-IV distorted octahedral geometry as evidenced from its crystal structure. The sugar appended complexes show significantly higher uptake into the cancer cells compared to their normal analogues. The complexes are remarkably photocytotoxic in visible light (400-700 nm) giving an IC50 value of <5 mu M in HeLa, HaCaT and MCF-7 cells with no significant dark toxicity. The green emission of the complexes was used for cellular imaging. Predominant cytosolic localization of the complexes 1-4 to a lesser extent into the nucleus was evidenced from confocal imaging. The complexes as strong binders of calf thymus DNA displayed photocleavage of supercoiled pUC19 DNA in red light by generating (OH)-O-center dot radicals as the ROS. The cell death is via an apoptotic pathway involving the ROS. Binding to the VO2+ moiety has resulted in stability against any hydrolytic degradation of curcumin along with an enhancement of its photocytotoxicity.

Synthesis, X-ray structure and in vitro cytotoxicity studies of Cu(I/II) complexes of thiosemicarbazone: special emphasis on their interactions with DNA

4-(p-X-phenyl)thiosemicarbazone of napthaldehyde {where X = Cl (HL1) and X = Br (HL2)}, thiosemicarbazone of quinoline-2-carbaldehyde (HL3) and 4-(p-fluorophenyl) thiosemicarbazone of salicylaldehyde (H2L4) and their copper(I) {Cu(HL1)(PPh3)(2)Br]center dot CH3CN (1) and Cu(HL2)(PPh3)(2)Cl]center dot DMSO (2)} and copper(II) {((Cu2L2Cl)-Cl-3)(2)(mu-Cl)(2)]center dot 2H(2)O (3) and Cu(L-4)(Py)] (4)} complexes are reported herein. The synthesized ligands and their copper complexes were successfully characterized by elemental analysis, cyclic voltammetry, NMR, ESI-MS, IR and UV-Vis spectroscopy. Molecular structures of all the Cu(I) and Cu(II) complexes have been determined by X-ray crystallography. All the complexes (1-4) were tested for their ability to exhibit DNA-binding and - cleavage activity. The complexes effectively interact with CT-DNA possibly by groove binding mode, with binding constants ranging from 10(4) to 10(5) M-1. Among the complexes, 3 shows the highest chemical (60%) as well as photo-induced (80%) DNA cleavage activity against pUC19 DNA. Finally, the in vitro antiproliferative activity of all the complexes was assayed against the HeLa cell line. Some of the complexes have proved to be as active as the clinical referred drugs, and the greater potency of 3 may be correlated with its aqueous solubility and the presence of the quinonoidal group in the thiosemicarbazone ligand coordinated to the metal.