From Rational Design of Drug Crystals to Understanding of Nucleic Acid Structures: Lamivudine Duplex

A DNA-like duplex of nucleosides is probable to exist even without the 5`-phosphate groups needed to assemble the chain backbone. However, double-stranded helical structures of nucleosides are unknown. Here, we report a duplex of nucleoside analogs that is spontaneously assembled due to stacking of the neutral and protonated molecules of lamivudine, a nucleoside reverse transcriptase inhibitor (NTRI) widely used in anti-HIV drug combinatory medication. The left-handed lamivudine duplex has features similar to those of i-motif DNA, as the face-to-face base stacking and the helix rise per base pair. Furthermore, the protonation pattern on alternate bases expected for it DNA-like duplex stabilized by pairing of neutral and protonated cytosine fragments was observed for the first time in the lamivudine double-stranded helix. This structure demonstrates that hydrogen bonds can substitute for covalent phosphodiester linkage in the stabilization of the duplex backbone. This interesting example of spontaneous molecular self-organization indicates that the 5`-phosphate group could not be a requirement for duplex assembly.

Descriptor-and fragment-based QSAR models for a series of Schistosoma mansoni purine nucleoside inhibitors

The enzyme purine nucleoside phosphorylase from Schistosoma mansoni (SmPNP) is an attractive molecular target for the treatment of major parasitic infectious diseases, with special emphasis on its role in the discovery of new drugs against schistosomiasis, a tropical disease that affects millions of people worldwide. In the present work, we have determined the inhibitory potency and developed descriptor- and fragment-based quantitative structure-activity relationships (QSAR) for a series of 9-deazaguanine analogs as inhibitors of SmPNP. Significant statistical parameters (descriptor-based model: r² = 0.79, q² = 0.62, r²pred = 0.52; and fragment-based model: r² = 0.95, q² = 0.81, r²pred = 0.80) were obtained, indicating the potential of the models for untested compounds. The fragment-based model was then used to predict the inhibitory potency of a test set of compounds, and the predicted values are in good agreement with the experimental results

Adaptive Immunity against Leishmania Nucleoside Hydrolase Maps Its C-Terminal Domain as the Target of the CD4+ T Cell-Driven Protective Response

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73 +/- 12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-gamma secretion, ratios of IFN-gamma/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNF alpha/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-gamma/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

Purine nucleoside phosphorylase from Schistosoma mansoni in complex with ribose-1-phosphate

Schistosomes are blood flukes which cause schistosomiasis, a disease affecting approximately 200 million people worldwide. Along with several other important human parasites including trypanosomes and Plasmodium, schistosomes lack the de novo pathway for purine synthesis and depend exclusively on the salvage pathway for their purine requirements, making the latter an attractive target for drug development. Part of the pathway involves the conversion of inosine (or guanosine) into hypoxanthine (or guanine) together with ribose-1-phosphate (R1P) or vice versa. This inter-conversion is undertaken by the enzyme purine nucleoside phosphorylase (PNP) which has been used as the basis for the development of novel anti-malarials, conceptually validating this approach. It has been suggested that, during the reverse reaction, R1P binding to the enzyme would occur only as a consequence of conformational changes induced by hypoxanthine, thus making a binary PNP-R1P complex unlikely. Contradictory to this statement, a crystal structure of just such a binary complex involving the Schistosoma mansoni enzyme has been successfully obtained. The ligand shows an intricate hydrogen-bonding network in the phosphate and ribose binding sites and adds a further chapter to our knowledge which could be of value in the future development of selective inhibitors.

Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl(2), 20% PEG 3350. Data were collected to 3.5 angstrom resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 angstrom. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease.

Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme from Schistosoma mansoni

Schistosomes are unable to synthesize purines de novo and depend exclusively on the salvage pathway for their purine requirements. It has been suggested that blockage of this pathway could lead to parasite death. The enzyme purine nucleoside phosphorylase (PNP) is one of its key components and molecules designed to inhibit the low-molecular-weight (LMW) PNPs, which include both the human and schistosome enzymes, are typically analogues of the natural substrates inosine and guanosine. Here, it is shown that adenosine both binds to Schistosoma mansoni PNP and behaves as a weak micromolar inhibitor of inosine phosphorolysis. Furthermore, the first crystal structures of complexes of an LMW PNP with adenosine and adenine are reported, together with those with inosine and hypoxanthine. These are used to propose a structural explanation for the selective binding of adenosine to some LMW PNPs but not to others. The results indicate that transition-state analogues based on adenosine or other 6-amino nucleosides should not be discounted as potential starting points for alternative inhibitors.

Influence of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity on Trypanosoma cruzi Infectivity and Virulence

Background: The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models. Methodology/Principal Findings: We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites. Conclusions/Significance: Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Sigma phase morphologies in cast and aged super duplex stainless steel

Solution annealed and water quenched duplex and super duplex stainless steels are thermodynamically metastable systems at room temperature. These systems do not migrate spontaneously to a thermodynamically stable condition because an energy barrier separates the metastable and stable states. However, any heat input they receive, for example through isothermal treatment or through prolonged exposure to a voltaic arc in the welding process, cause them to reach a condition of stable equilibrium which, for super duplex stainless steels, means precipitation of intermetallic and carbide phases. These phases include the sigma phase, which is easily identified from its morphology, and its influence on the material`s impact strength. The purpose of this work was to ascertain how 2-hour isothermal heat treatments at 920 degrees C and 980 degrees C affect the microstructure of ASTM A890/A890M GR 6A super duplex stainless steel. The sigma phase morphologies were found to be influenced by these two aging temperatures, with the material showing a predominantly lacy microstructure when heat treated at 920 degrees C and block-shaped when heat treated at 980 degrees C. (C) 2009 Elsevier Inc. All rights reserved.

Microstructural characteristics and corrosion behavior of a super duplex stainless steel casting

The machining of super duplex stainless steel castings is usually complicated by the difficulty involved in maintaining the dimensional tolerances required for given applications. Internal stresses originating from the solidification process and from subsequent heat treatments reach levels that exceed the material`s yield strength, promoting plastic strain. Stress relief heat treatments at 520 degrees C for 2 h are an interesting option to solve this problem, but because these materials present a thermodynamically metastable condition, a few precautions should be taken. The main objective of this work was to demonstrate that, after solution annealing at 1130 degrees C and water quenching, stress relief at 520 degrees C for 2 h did not alter the duplex microstructure or impair the pitting corrosion resistance of ASTM A890/A890M Grade 6A steel. This finding was confirmed by microstructural characterization techniques, including light optical and scanning electron microscopy, and X-ray diffraction. Corrosion potential measurements in synthetic sea water containing 20,000 ppm of chloride ions were also conducted at three temperatures: 5 degrees C, 25 degrees C and 60 degrees C. (c) 2008 Elsevier Inc. All rights reserved.

Ultrasound, eddy current and magnetic Barkhausen noise as tools for sigma phase detection on a UNS S31803 duplex stainless steel

Sigma phase is a deleterious one which can be formed in duplex stainless steels during heat treatment or welding. Aiming to accompany this transformation, ferrite and sigma percentage and hardness were measured on samples of a UNS S31803 duplex stainless steel submitted to heat treatment. These results were compared to measurements obtained from ultrasound and eddy current techniques, i.e., velocity and impedance, respectively. Additionally, backscattered signals produced by wave propagation were acquired during ultrasonic inspection as well as magnetic Barkhausen noise during magnetic inspection. Both signal types were processed via a combination of detrended-fluctuation analysis (DFA) and principal component analysis (PCA). The techniques used were proven to be sensitive to changes in samples related to sigma phase formation due to heat treatment. Furthermore, there is an advantage using these methods since they are nondestructive. (C) 2010 Elsevier B.V. All rights reserved.

Surface integrity analysis in the super duplex stainless steel ASTM-A890 after machining

The purpose of this paper was to study the main effects of the turning in the superficial integrity of the duplex stainless steel ASTM A890-6A. The tests were conducted on a turning centre with carbide tools and the main entrances variables were: tool material class, feed rate, cutting depth, cutting speed and cutting fluid utilisation. The answers were analysed: microstructural analysis by optical microscopy and x-ray diffraction, cutting forces measurements by a piezoelectric dynamometer, surface roughness, residual stress by x-ray diffraction technique and the microhardness measurements. The results do not show any changes in the microstructural of the material, even when the greater cutting parameters were used. The smaller feed rate (0.1 mm/v), smaller cutting speed (110 m/min) and the greater cutting depth (0.5 mm) provided the smaller values for the tensile residual stress, the smaller surface roughness and the greater microhardness.

Microstructure and texture of duplex stainless steel after melt-spinning processing

The microstructure and texture of melt-spun UNS S31803 (DIN W. Nr. 1. 4462) duplex stainless steel were analyzed after casting and solution treatment. The cast ribbons contained austenite (gamma) and ferrite (alpha or delta) with roughly equal compositions. The alpha and gamma had < 100 > and < 110 > partial fiber textures, respectively. After solution treatment, the texture was maintained, the amount of gamma phase increased, and the alloying elements were partitioned as expected, according to whether they were ferrite or austenite stabilizers. (c) 2006 Elsevier Inc. All rights reserved.

Corrosion Versus Mechanical Tests for Indirect Detection of Alpha Prime Phase in UNS S32520 Super Duplex Stainless Steel

Alpha prime formation leads to material embrittlement and deterioration of corrosion resistance. In the present study, the mechanical and corrosion behavior of super duplex stainless steel UNS S32520 aged at 475 degrees C from 0.5 h to 1,032 h was evaluated using microhardness measurements, Charpy impact tests, electrochemical impedance spectroscopy, and cyclic polarization curves. The sensibility of these tests to the effects of alpha prime phase was investigated. The microhardness test showed a gradual increase in hardness with aging time, whereas the impact tests revealed losses of about 80% in the energy absorption capacity for the material aged for 12 h in comparison with the solution-annealed samples. The most responsive analysis was the impact test, which indirectly revealed the presence of this deleterious phase in samples aged for 0.5 h. The electrochemical impedance spectroscopy and polarization tests were not highly sensitive to the alpha prime phase unless these are present in large amounts in the stainless steel.

Improvement of the cavitation erosion resistance of UNS S31803 stainless steel by duplex treatment

A duplex surface treatment consisting of High Temperature Gas Nitriding (HTGN) followed by Low Temperature Plasma Nitriding (LTPN) was carried out in an UNS S31803 duplex stainless steel. The HTGN treatment was intended to produce a relatively thick and hard fully austenitic layer giving mechanical support to the thinner and much harder expanded austenite layer. HTGN was performed at 1200 degrees C for 3 h, in a 0.1 MPa N(2) atmosphere while LTPN, was carried out in a 75% N(2) + 25% H(2) atmosphere, at 400 degrees C for 12 h, under a 250 Pa pressure, and 450 V. An expanded austenite gamma(N) layer, 2.3 mu m thick, 1500 HVO.025 hard, was formed on top of a 100 mu m thick, 330 HV 0.1 hard, fully austenitic layer, containing 0.9 wt% N. For comparison purposes LTPN was carried out with UNS S30403 stainless steel specimens obtaining a 4.0 mu m thick, 1500 HV 0.025 hard, expanded austenite layer formed on top of a fully austenitic matrix having 190 HV 0.1. The nitrided specimens were tested in a 20 kHz vibratory cavitation-erosion testing equipment. Comparison between the duplex treated UNS S31803 steel and the low temperature plasma nitrided UNS S30403 steel, resulted in incubation times almost 9 times greater. The maximum cavitation wear rate of the LTPN UNS S30403 was 5.5 g/m(2)h, 180 times greater than the one measured for the duplex treated UNS S31803 steel. The greater cavitation wear resistance of the duplex treated UNS S31803 steel, compared to the LTPN treated UNS S30403 steel was explained by the greater mechanical support the fully austenitic, 330 HV 0.1 hard, 100 mu m layer gives to the expanded austenite layer formed on top of the specimen after LTPN. A strong crystallographic textured surface, inherited from the fully austenitic layer formed during HTGN, with the expanded austenite layer showing {101} crystallographic planes//surface contributed also to improve the cavitation resistance of the duplex treated steel. (C) 2010 Elsevier B.V. All rights reserved.

Intragranular formation of austenite during delta ferrite decomposition in a duplex stainless steel

A Fe-22.5%Cr-4.53%Ni-3.0%Mo duplex stainless steel was solution treated at 1,325 A degrees C for 1 h, quenched in water and isothermally treated at 900 A degrees C for 5,000 s. The crystallography of austenite was studied using EBSD technique. Intragranular austenite particles formed from delta ferrite are shown to nucleate on inclusions, and to be subdivided in twin-related sub-particles. Intragranular austenite appears to have planar-only orientation relationships with the ferrite matrix, close to Kurdjumov-Sachs and Nishyiama-Wassermann, but not related to a conjugate direction. Samples treated at 900 A degrees C underwent sparse formation of sigma phase and pronounced growth of elongated austenite particles, very similar to acicular ferrite.