Discovery and optimization of piperidyl benzamide derivatives as a novel class of 11β-HSD1 inhibitors

Discovery and optimization of a piperidyl benzamide series of 11β-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which features improved properties over the cyclohexyl benzamide derivatives.     

The in vivo effect of benzamide and phenobarbital on liver enzymes: poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.     

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

We have determined the ability of UV_254nm-irradiated murine lymphoma cells to adapt their NAD⁺ metabolism to the increased NAD⁺ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV_254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV_254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD⁺-utilizing enzyme poly(ADP-ribose) polymerase, NAD⁺ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV_254nm irradiation, a dramatic transient increase of NAD⁺ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV_254nm irradiation, the NAD⁺ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD⁺ metabolism to UV_254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD⁺ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells.     

Atropisomeric amides: Achiral ligands with chiral conformations

A new class of achiral ligands with atropisomeric conformations has been coordinated to titanium(IV). The ligands are ortho-hydroxy benzamide derivatives which are deprotonated on reaction with titanium tetraisopropoxide to furnish Ti(L)(2)(O-iPr)(2) complexes (L=ortho-phenoxy benzamide). In these octahedral titanium compounds, the ortho-phenoxy benzamide ligands chelate to titanium, bonding through the phenoxide oxygen and the amide carbonyl oxygen. The benzamide ligands adopt atropisomeric conformations with an angle between the aryl and amide groups of approximately 35 degrees. The ligand precursor, ligand, and titanium complexes have been characterized by X-ray crystallography. Only one diastereomer of each titanium complex was observed in the solid state structures.     

Why Do Child-Directed Interactions Support Imitative Learning in Young Children?

Child-directed cues support imitation of novel actions at 18 months, but not at two years of age. The current studies explore the mechanisms that underlie the propensity that children have to copy others at 18 months, and how the value of child-directed communication changes over development. We ask if attentional allocation accounts for children''s failure to imitate observed actions at 18 months, and their success at two years of age, and we explore the informational value child-directed contexts may provide across ontogeny. Eighteen-month-old (Study 1) and two-year-old (Study 2) children viewed causally non-obvious actions performed by child-directed (Study 1 & 2), observed (Study 1 & 2), or non-interactive (Study 2) actors, and their visual attention and imitative behaviors were assessed. Results demonstrated that child-directed contexts supported imitative learning for 18-month-old children, independent of their effects on proximal attention. However, by two years of age, neither directness nor communication between social partners was a necessary condition for supporting social imitation. These findings suggest that developmental changes in children''s propensity to extract information from observation cannot be accounted for by changes in children''s interpretation of what counts as child-directed information, and are likely not due to changes in how children allocate attention to observed events.     

Methamphetamine (METH) causes reactive gliosis in vitro: Attenuation by the ADP-ribosylation (ADPR) inhibitor,benzamide

We examined the effects of methamphetamine (METH) in an in vitro model of rat fetal mesencephalic cells. METH causes loss of dopamine (DA) cells and neuronal process degeneration. In addition, the drug causes an increase in reactive gliosis as shown by the number of cells that stain for and by the intensity of staining with a glial fibrillary acidic protein (GFAP) antibody. Co-incubation of METH-treated cells with benzamide, which is a known inhibitor of ADP-ribosylation (ADPR), attenuated METH effects on both DA and glial cells. However, the effects of benzamide were somewhat more prominent on the glial cells. These results suggest that ADP-ribosylation may play a very important role in the development of reactive gliosis after the administration of neurotoxic agents.     

Exploring the cocrystallization potential of urea and benzamide

The cocrystallization landscape of benzamide and urea interacting with aliphatic and aromatic carboxylic acids was studied both experimentally and theoretically. Ten new cocrystals of benzamide were synthesized using an oriented samples approach via a fast dropped evaporation technique. Information about types of known bi-component cocrystals augmented with knowledge of simple binary eutectic mixtures was used for the analysis of virtual screening efficiency among 514 potential pairs involving aromatic carboxylic acids interacting with urea or benzamide. Quantification of intermolecular interaction was achieved by estimating the excess thermodynamic functions of binary liquid mixtures under supercooled conditions within a COSMO-RS framework. The smoothed histograms suggest that slightly more potential pairs of benzamide are characterized in the attractive region compared to urea. Finally, it is emphasized that prediction of cocrystals of urea is fairly direct, while it remains ambiguous for benzamide paired with carboxylic acids. The two known simple eutectics of urea are found within the first two quartiles defined by excess thermodynamic functions, and all known cocrystals are outside of this range belonging to the third or fourth quartile. On the contrary, such a simple separation of positive and negative cases of benzamide miscibility in the solid state is not observed. The difference in properties between urea and benzamide R2,2(8) heterosynthons is also documented by alterations of substituent effects. Intermolecular interactions of urea with para substituted benzoic acid analogues are stronger compared to those of benzamide. Also, the amount of charge transfer from amide to aromatic carboxylic acid and vice versa is more pronounced for urea. However, in both cases, the greater the electron withdrawing character of the substituent, the higher the binding energy, and the stronger the supermolecule polarization via the charge transfer mechanism.     

DNA-strand breaks associated with halogenated pyrimidine incorporation

The alkaline elution of bromodeoxyuridine-containing (BrdUrd) DNA and chlorodeoxyuridine-containing ( CldUrd ) DNA was studied in two CHO lines, the parental AA8 and a mutant line, EM9 , which has a defect in repairing strand breaks and a 12-fold elevated baseline frequency of SCE. BrdUrd-DNA was found to have alkali-labile sites as well as direct breaks, neither of which were increased significantly by prior treatment of AA8 cells with an inhibitor (benzamide) or poly(adenosine diphosphoribose) polymerase. CldUrd -DNA, which gives higher frequencies of SCEs than BrdUrd-DNA, had more strand breaks than BrdUrd-DNA in AA8 cells after treatment with benzamide, while without benzamide there was no difference. The accumulation of breaks in CldUrd -DNA by benzamide was shown to occur rapidly, to reach a maximum by 90 min, and to be readily reversible after benzamide removal. Under all conditions, EM9 cells had more strand breaks than AA8 . These observed differences in strand breaks were not due to differences in incorporation efficiencies. For the different halogenated pyrimidines and cell types, there was a good correlation between the number of strand breaks and reduction in plating efficiencies.     

Men report stronger attraction to femininity in women's faces when their testosterone levels are high

Many studies have shown that women's judgments of men's attractiveness are affected by changes in levels of sex hormones. However, no studies have tested for associations between changes in levels of sex hormones and men's judgments of women's attractiveness. To investigate this issue, we compared men's attractiveness judgments of feminized and masculinized women's and men's faces in test sessions where salivary testosterone was high and test sessions where salivary testosterone was relatively low. Men reported stronger attraction to femininity in women's faces in test sessions where salivary testosterone was high than in test sessions where salivary testosterone was low. This effect was found to be specific to judgments of opposite-sex faces. The strength of men's reported attraction to femininity in men's faces did not differ between high and low testosterone test sessions, suggesting that the effect of testosterone that we observed for judgments of women's faces was not due to a general response bias. Collectively, these findings suggest that changes in testosterone levels contribute to the strength of men's reported attraction to femininity in women's faces and complement previous findings showing that testosterone modulates men's interest in sexual stimuli.     

A benzamide‐dependent ftsZ mutant reveals residues crucial for Z‐ring assembly

In almost all bacteria, cell division is co‐ordinated by the essential tubulin homologue FtsZ and represents an attractive but as yet unexploited target for new antibiotics. The benzamides, e.g. PC190723, are potent FtsZ inhibitors that have the potential to yield an important new class of antibiotic. However, the evolution of resistance poses a challenge to their development. Here we show that a collection of PC190723‐resistant and ‐dependent strains of Staphylococcus aureus exhibit severe growth and morphological defects, questioning whether these ftsZ mutations would be clinically relevant. Importantly, we show that the most commonly isolated substitution remains sensitive to the simplest benzamide 3‐MBA and likely works by occluding compound binding. Extending this analysis to Bacillus subtilis, we isolated a novel benzamide‐dependent strain that divides using unusual helical division events. The ftsZ mutation responsible encodes the substitution of a highly conserved residue, which lies outside the benzamide‐binding site and forms part of an interface between the N‐ and C‐terminal domains that we show is necessary for normal FtsZ function. Together with an intragenic suppressor mutation that mimics benzamide binding, the results provide genetic evidence that benzamides restrict conformational changes in FtsZ and also highlights their utility as tools to probe bacterial division.     

Observations of aspergillosis in the brains of turkey poults using different histopathological staining techniques

Different staining methods were evaluated for studying aspergillosis of the central nervous system (CNS). The pathological changes and fungal elements in cerebrum and cerebellum of 17 turkey poults with aspergillosis were examined and described. Tissue sections were stained with hematoxylin-eosin (HE), Klüver-Barrera's and Grocott's methods, and periodic acid-Schiff (PAS). Focal granulomatous reactions with central necrosis were observed in the HE stained slides. Fungal hyphae were easily demonstrated using Grocott's method and PAS. These two methods, however, were not suitable for describing detailed histopathological changes. The Klüver-Barrera method was used to demonstrate the neural tissue reaction. Neurons were found to be sensitive to aspergillosis, in contrast to glial cells that showed fewer pathological changes. The fungal elements were clearly visible with the Klüver-Barrera method, resulting in better information about the interactions of neural tissue, the inflammatory response, and the fungus. The use of the Klüver-Barrera method for this purpose has not been documented previously.     

Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo

The ADP-ribosyl moiety of NAD+ is consumed in reactions catalyzed by three classes of enzymes: poly(ADP-ribose) polymerase, protein mono(ADP-ribosyl)transferases, and NAD+ glycohydrolases. In this study, we have evaluated the selectivity of compounds originally identified as inhibitors of poly(ADP-ribose) polymerase on members of the three classes of enzymes. The 50% inhibitory concentration (IC50) of more than 20 compounds was determined in vitro for both poly(ADP-ribose) polymerase and mono(ADP-ribosyl)transferase A in an assay containing 300 microM NAD+. Of the compounds tested, benzamide was the most potent inhibitor of poly(ADP-ribose) polymerase with an IC50 of 3.3 microM. The IC50 for benzamide for mono(ADP-ribosyl)transferase A was 4.1 mM, and similar values were observed for four additional cellular mono(ADP-ribosyl)transferases. The IC50 for NAD+ glycohydrolase for benzamide was approximately 40 mM. For seven of the best inhibitors, inhibition of poly(ADP-ribose) polymerase in intact C3H1OT1/2 cells was studied as a function of the inhibitor concentration of the culture medium, and the concentration for 50% inhibition (culture medium IC50) was determined. Culture medium IC50 values for benzamide and its derivatives were very similar to in vitro IC50 values. For other inhibitors, such as nicotinamide, 5-methyl-nicotinamide, and 5-bromodeoxyuridine, culture medium IC50 values were 3-5-fold higher than in vitro IC50 values. These results suggest that micromolar levels of the benzamides in the culture medium should allow selective inhibition of poly(ADP-ribose) metabolism in intact cells. Furthermore, comparative quantitative inhibition studies should prove useful for assigning the biological effects of these inhibitors as an effect on either poly(ADP-ribose) or mono(ADP-ribose) metabolism.     

Quantitative historical change in bumblebee (Bombus spp.) assemblages of red clover fields

Background

Flower visiting insects provide a vitally important pollination service for many crops and wild plants. Recent decline of pollinating insects due to anthropogenic modification of habitats and climate, in particular from 1950''s onwards, is a major and widespread concern. However, few studies document the extent of declines in species diversity, and no studies have previously quantified local abundance declines. We here make a quantitative assessment of recent historical changes in bumblebee assemblages by comparing contemporary and historical survey data.

Methodology/Principal Findings

We take advantage of detailed, quantitative historical survey data from the 1930''s on bumblebee (Bombus spp.) abundances and species composition in red clover (Trifolium pratense) fields, an important floral resource and an attractant of all bumblebee species. We used the historical survey data as a pre-industrialization baseline, and repeated the same sampling protocol at nearly the same localities at present, hence setting up a historical experiment. We detected historical changes in abundances (bees/m²) of both workers (the “pollinatory units”) and queens (effective population size), in addition to species composition. In particular, long-tongued bumblebee species showed consistent and dramatic declines in species richness and abundances throughout the flowering season of red clover, while short-tongued species were largely unaffected. Of 12 Bombus species observed in the 1930''s, five species were not observed at present. The latter were all long-tongued, late-emerging species.

Conclusions/Significance

Because bumblebees are important pollinators, historical changes in local bumblebee assemblages are expected to severely affect plant reproduction, in particular long-tubed species, which are pollinated by long-tongued bumblebees.     

Synthesis,in Silico Study and Biological Evaluation of N-(Benzothiazol/Thiazol-2-yl)benzamide Derivatives as Quorum Sensing Inhibitors against Pseudomonas aeruginosa

The development of bacterial resistance to chemical therapy poses a severe danger to efficacy of treating bacterial infections. One of the key factors for resistance to antimicrobial medications is growth of bacteria in biofilm. Quorum sensing (QS) inhibition was created as an alternative treatment by developing novel anti-biofilm medicines. Cell-cell communication is impeded by QS inhibition, which targets QS signaling pathway. The goal of this work is to develop newer drugs that are effective against Pseudomonas aeruginosa by decreasing QS and acting as anti-biofilm agents. In this investigation, N-(benzo[d]thiazol-2-yl)benzamide/N-(thiazol-2-yl)benzamide derivatives 3a-h were designed and synthesized in good yields. Further, molecular docking analyses revealed that binding affinity values were founded −11.2 to −7.6 kcal/mol that were moderate to good. The physicochemical properties of these prepared compounds were investigated through in-silico method. Molecular dynamic simulation was also used to know better understanding of stability of the protein and ligand complex. Comparing N-(benzo[d]thiazol-2-yl)benzamide 3a to salicylic acid (4.40±0.10) that was utilised as standard for quorum sensing inhibitor, the anti-QS action was found greater for N-(benzo[d]thiazol-2-yl)benzamide 3a (4.67±0.45) than salicylic acid (4.40±0.10). Overall, research results suggested that N-(benzo[d]thiazol-2-yl)benzamide/N-(thiazol-2-yl)benzamide derivatives 3a-h may hold to develop new quorum sensing inhibitors.     

Few changes in native Australian alpine plant morphology,despite substantial local climate change

Rapid evolution is likely to be an important mechanism allowing native species to adapt to changed environmental conditions. Many Northern Hemisphere species have undergone substantial recent changes in phenology and morphology. However, we have little information about how native species in the Southern Hemisphere are responding to climate change. We used herbarium specimens from 21 native alpine plant species in Kosciuszko National Park, Australia, to make over 1,500 measurements of plant size, leaf thickness, leaf mass per area, leaf shape, and leaf size across the last 126 years. Only two out of 21 species (9%) showed significant changes in any of the measured traits. The number of changes we observed was not significantly different to what we would expect by chance alone, based on the number of analyses performed. This lack of change is not attributable to methodology—an earlier study using the same methods found significant changes in 70% of species introduced to southeast Australia. Australia''s native alpine plants do not appear to be adapting to changed conditions, and because of the low elevation of Australia''s mountains, they do not have much scope for uphill migration. Thus, our findings suggest that Australia''s native alpine plants are at even greater risk in the face of future climate change than was previously understood.     

Effects of benzamide pretreatment on clastogenic adaptation of Vicia faba root-tip meristem cells to triethylenemelamine (TEM) and maleic hydrazide (MH)

Clastogenic adaptation to TEM or MH no longer occurred when benzamide, an inhibitor of nuclear ADP-ribosyltransferase (ADPRT), was applied prior to the low dose (conditioning) treatment which triggers this phenomenon. This may be indicative that inducible processes connected with ribosylation reactions are involved in the protective effects exerted by clastogenic adaptation. No increase by benzamide pretreatment was observed in the yield of metaphases with TEM- or MH-induced chromatid aberrations after conditioning and challenge treatment, respectively. High benzamide concentrations (1 h, 5 X 10(-3) M) exerted protective effects against TEM challenging but not against MH.     

Seasonal and annual variation in the diving behaviour of Hutton's shearwater (Puffinus huttoni)

ABSTRACT

With the development and implementation of tracking technology, we are now able to monitor the foraging behaviour of seabirds while at sea. Time-Depth Recorders (TDRs) were fitted to Hutton's shearwaters (Puffinus huttoni), an endangered endemic New Zealand species, to measure how diving behaviour varies over the breeding cycle. Hutton's shearwaters (～350?g) dive up to 339 times per day (average 68.8) at depths to 35?m (average 5.6?m), and for periods up to 60?s (average 19.2?s). Incubating birds dived deeper than birds feeding chicks, and a significant difference in diving depth and dive duration were detected at different times of the day. Neither dive frequency nor dive duration differed significantly between years, but there was some annual variation in dive depths. The temporal variation we observed in the diving behaviour of Hutton's shearwaters suggests they are likely to exploit different types of pelagic prey at different stages in their breeding cycle. With on-going changes in the marine environment, monitoring changes in feeding behaviour using TDRs may provide a way to assess environmental change and improve the conservation of this species.     

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labelsm-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl)-m-(fluorosulfonyl)benzamide] andm-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl)benzamide], which are sensitive to differences betweenα- andγ-thrombin, were quite similar to that ofα-thrombin. That is, no major conformational differences between meizothrombin(desF1) andα-thrombin were observed in this region of the extended active site. On the other hand,p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide],p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl)benzamide], andm-VII [N-[m-(fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl-piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility betweenα-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labelsp-IV,p-V, andm-VII, which corresponds to the apolar binding region, differs in conformation fromα-thrombin.