text:
Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.
Methodology/Principal Findings
The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar KD values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC50 <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC50 of 17 nM and was trypanocidal at 40 nM.
The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.
Author Summary
Chagas Disease is the leading cause of heart disease in Latin America and an emerging infection in Europe and North America. The clinical presentation of Chagas Disease arises from infection by the protozoan parasite Trypanosoma cruzi, which leads to progressive cardiomyopathy. No vaccine is available and chemotherapeutic options are limited to the drugs benznidazole and nifurtimox, which are used during the acute phase but may cause severe gastrointestinal and neurological side effects and are not commonly used in the chronic phase. Neither drug is approved by the FDA for use in the United States. The need for effective new therapy is urgent. A validated therapeutic target in T. cruzi is CYP51, an essential enzyme in the sterol biosynthesis pathway. We report results of high-throughput screening of small molecules directly against CYP51, confirmed by in vitro medium-throughput screening of the hits against T. cruzi-infected mammalian cells. We have identified a potent T. cruzi inhibitor as well as a diverse collection of low molecular weight hits with high affinity to CYP51. We have applied computational chemistry to relate CYP51 to other pharmacologic targets. This analysis allowed us to identify molecules already produced by pharmaceutical companies for future experimental testing against T. cruzi.
Citation: Gunatilleke SS, Calvet CM, Johnston JB, Chen C-K, Erenburg G, Gut J, et al. (2012) Diverse Inhibitor Chemotypes Targeting Trypanosoma cruzi CYP51. PLoS Negl Trop Dis 6(7): e1736. https://doi.org/10.1371/journal.pntd.0001736
Editor: Michael P. Pollastri, Northeastern University, United States of America
Received: March 23, 2012; Accepted: June 4, 2012; Published: July 31, 2012
Copyright: © Gunatilleke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health (NIH) RO1 Grant AI095437 (to L.M.P.) and The Sandler Foundation. Screening was supported by an Award from The Center of Emerging and Neglected Diseases (CEND) at University of California Berkeley (to L.M.P.). C.M.C. was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and FIOCRUZ; K.K.H.A. was supported by the QB3-Malaysia program; J.B.J. was supported by NIH R01 Grant AI74824 (to Prof. Ortiz de Montellano, University of California San Francisco). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The parasitic protozoon Trypanosoma cruzi, the etiological agent of Chagas Disease, is naturally transmitted by blood-sucking bugs of the subfamily Triatominae [1]. The range of clinically approved drugs for both the acute and chronic stage of the disease is limited and new medications are urgently needed [2]–[4]. The integrity of the parasite's cell membrane depends on specific endogenously synthesized 24-methylated sterols [5], membrane building blocks which cannot be entirely substituted by cholesterol scavenged from the host in the clinically relevant amastigote stage. The sterol biosynthesis pathway in T. cruzi is thus essential for the survival of the replicative intracellular amastigote stage in infected human hosts [5]–[7]. The similarity between membrane sterol requirements of T. cruzi and those of pathogenic fungi has led to the idea of repurposing anti-fungal azole and triazole drugs for Chagas Disease therapy. For example, posaconazole and ravuconazole target sterol 14α-demethylase (CYP51), the key enzyme in sterol biosynthesis, and therefore have potential as therapeutics [8]–[12]. Inhibitors of CYP51 deplete endogenous 24-methylated sterols in the intracellular amastigote stage, resulting in blebbing of the cell membrane, deterioration of internal membranes and finally in T. cruzi cell death [13], [14].
Commercial anti-fungal drugs of the "conazole" pedigree are derived from miconazole that was mainly used for topical treatment of fungal infections [15]. Posaconazole, the most recent member of this family to be marketed, is considered not only a valuable addition to the therapeutic armamentarium against systemic life-threatening fungal infections [16], [17], but also is a clinical trial candidate for Chagas Disease. The anti-chagasic potency of posaconazole has been demonstrated in an animal model of T. cruzi infection [9], [18], [19] and in humans [12]. However, potential issues of drug resistance, cost and safety mar the prospective utility of azole drugs. To broaden the range of CYP51 inhibitors that could be used most efficaciously to treat patients with Chagas Disease, we employed an approach that relies on directly probing the CYP51 active site with diverse compounds to define the chemical and structural space accessible to inhibitors. This approach is based on advances in high throughput screening, medicinal chemistry, structural biology, and cheminformatics, and with it we have identified a broad selection of promising molecular scaffolds.
As a member of the P450 family, CYP51 shares the common spectral property of P450 enzymes in which the ferric Fe Soret band is shifted following the displacement (type I shift) or replacement (type II shift) of a water molecule, a weak axial ligand, by a stronger one, usually one containing a nitrogen aliphatic or aromatic group [20]. The concentration-dependence of the spectral changes allows the binding affinities of the ligand to be estimated. A spectroscopic assay utilizing these properties has been routinely used to explore P450-ligand interactions in low-throughput format. Three major advantages of the assay that make it attractive for high-throughput applications are its simplicity, applicability to poorly characterized enzymes, and universality, as it can be easily adapted for analysis of any CYP obtainable in soluble form. The main drawbacks of the assay, however, are the relatively low-throughput and inherently low sensitivity of UV-vis absorption spectroscopy, which requires micromolar protein concentrations for screening. This in turn leads potentially to interference with test compound optical properties or solubility and to increased numbers of false-positive and false-negative hits [21]. Despite these disadvantages, a high throughput screening (HTS) assay based on the shift of the heme iron Soret band in response to small molecule binding has been successfully applied to CYP51's counterpart in Mycobacterium tuberculosis [22], ultimately resulting in identification of a potent T. cruzi inhibitor [14], [23] whose drug-like properties for Chagas Disease are currently being optimized.
The successful development of a structurally characterized, high expression recombinant T. cruzi CYP51 [24] has led to the establishment of an experimental platform for direct high-throughput exploration of the CYP51 active site capable of accommodating larger and more diverse libraries. Here we describe how a library of ∼104,000 small molecules was reduced to 185 structurally diverse positive hits with estimated KD values in the nanomolar range. Cross-validation of hits against T. cruzi parasites cultured in mammalian cells revealed 57 hits with anti-T. cruzi activity defined by EC50 values below 10 µM. While a region of structural similarity among the high affinity hits is dictated by the requirement for an aromatic heterocycle capable of coordinating to the heme iron, other parts of the molecules are not so constrained, allowing for unprecedented structural diversity among potential hits and opening new horizons for medicinal chemistry exploration of CYP51.
Materials and Methods
CYP51 target expression and purification
A previously constructed expression vector [24] was used to generate the recombinant T. cruzi CYP51 described in this work. The CYP51 utilized in the HTS screens had the first 21 residues upstream of K22 replaced with the fragment MAKKKKK and was His6-tag-engineered at the C-terminus. Protein was purified as described elsewhere [24] and was observed by means of 12% SDS-PAGE to be virtually homogeneous. Chromatographic fractions containing CYP51 in 20 mM potassium phosphate, pH 8.2, 10% glycerol, 0.5 mM EDTA, and 1 mM DTT and 200 mM NaCl were combined, concentrated and stored at −80°C. As expected, the enzyme solution displayed an absorbance peak at 420 nm, with a 449 nm peak appearing after reduction with sodium dithionite and bubbling with CO [24]. The concentration of functional P450 was determined from difference spectra using the extinction coefficient ε450–490 = 91000 M−1 cm−1 [25]. Purified CYP51 was shown to be stable under screening conditions at room temperature for up to 4 hours.
High-throughput assay overview
The compound library of the UCSF Small Molecule Discovery Center (SMDC), consisting of 103,986 diverse small molecules purchased from ChemBridge (San Diego, California), ChemDiv (San Diego, California) and SPECS (Delft, Netherlands) was screened against recombinant T. cruzi CYP51 in 384-well format using the protocol developed for M. tuberculosis CYP51 and described elsewhere [21]. The following modifications were introduced in the protocol to improve signal-to-noise ratio, throughput and reproducibility of measurements. First, the working protein concentration was increased to 2 µM and compounds were screened at 12.5 µM using two-wavelength detection. In this primary screen, plate reading was performed at 425 nm and 390 nm, corresponding to the maxima and minima of the difference spectra resulting from type II binding in the CYP51 active site (Figure 1). Type II inhibitors are characterized by their coordination bond to the heme iron, the single strongest interaction contributing to target affinity. Next, to account for organic solvent effects, protein background and the optical properties of test compounds, plates were prepared in duplicate, with target protein loaded in only a single set. After being read and processed separately, values generated from the protein-minus reference plate were subtracted from the corresponding values in the protein-plus test plate, yielding absorbance contributions due solely to the protein-compound interactions (Figure 1). Finally, to validate two-wavelength positive hits, four serial dilutions ranging from 2 µM to 12.5 µM were tested for a subset of positive hits using spectral detection mode, the spectra being recorded between 350 nm and 500 nm.
T. cruzi CYP51 target is shown clipped by plane through the substrate binding tunnel (top, center). Type II difference spectrum is recorded for 1 µM CYP51 at saturated concentration of reference compound (top, right). Screen plates were prepared in duplicate, A and B, with target protein loaded only in A set (left). HTS scatter plot shows absorbance differences of test compounds (ΔA = A425−A390) measured using the two-wavelength detection mode; each point represents a single test compound (bottom, center). Overlapped spectra are shown for a sub-micromolar hit re-evaluated in spectral mode at four serial dilutions (bottom, right).
Plate design, preparation and reading
All measurements were conducted in 100 mM potassium phosphate, pH 7.5, supplemented with 10% glycerol and, according to the plate layout shown in Figure 1, with dimethyl sulfoxide (DMSO) (columns 1 and 2), reference compound (columns 23–24) or individual test compounds (columns 3 through 22) at required concentrations. First, buffer alone (reference plates) or buffer containing 2 µM CYP51 (sample plates) was distributed in 80 µl aliquots using a Wellmate liquid dispenser (Thermo Fisher Matrix). Then test compounds, reference or DMSO were added in 1-µl aliquots using the automated 384-well head pipettor (Biomek FXP, Beckman Coulter) fitted with P20 tips (Fluotics Inc.). The compound {α-[(4-methylcyclohexyl)carbonyl amino]-N-4-pyridinyl-1H-indole-3-propanamide} purchased from ChemDiv (# C155-0123) (San Diego, California), referenced previously as LP-10 [14], [23], [26], was used as a type II reference. The compound and the reference stock solutions were prepared in DMSO at concentrations 80 times above working concentrations. DMSO alone was used as needed to hold DMSO concentration constant at 1.25% across the wells. Once prepared, plates were vortexed for 3 min, 800 rpm in a titer plate shaker (Lab-line Instrument, Inc., Pasadena, TX) and pulse-centrifuged for 10 sec, 1000 rpm (250 g) (Marathon 3000R, Fisher Scientific) to remove bubbles. Using a multimode plate reader (SpectraMax M5, Molecular Devices), the absorbance was recorded at 425 nm and 390 nm in two-wavelength mode, or at between 350 nm to 500 nm in 10 nm increments (16 measurements total/compound) in spectral mode, with 6 reads per well.
Analysis of plate readings
Data were manipulated and analyzed using Microsoft Office Excel 2007. Values from each pair of test and reference plates were read and exported to the pre-programmed Excel template and processed using the algorithm shown in Table S1. First, each plate was processed separately to account for the effects of DMSO and protein background. In this step absorbance values for each individual wavelength were averaged across all 32 wells in columns 1 and 2. The resulting value was subtracted from the absorbance measured at the same wavelengths for each individual compound (columns 3 through 22) or reference (columns 23–24). Next, the corrected absorbance values in the reference plate (Table S1B) were subtracted from those of corresponding wells in the test plate (Table S1A), yielding the absorbance contributions at each wavelength solely due to protein-compound interactions. In two-wavelength mode, ΔA = A425−A390 was calculated for each compound and plotted against the corresponding well (Figure 1). Due to the Soret band shifting red, ΔA is a positive value for type II hits. In spectral mode, corrected absorbance values were directly plotted against wavelength. The resulting difference binding spectra were overlaid and inspected visually to eliminate false-positive hits and to rank the remaining compounds in the order of their affinities to the CYP51 target (Figure 2).
Absorbance was monitored at four serial dilutions for each tested compound. (A) Lack of characteristic spectra-score 0; (B) concentration dependence with no spectra overlap – score 1; (C) any two spectra overlap-score 2; (D) any three spectra overlap-score 3; (E) three highest concentrations overlap-score 4; (F) all four spectra overlap-score 5.
Ranking positive hits in spectral mode
For ranking purposes, compounds were scored according to the appearance of their spectra. A score of 0 was assigned to false-positive compound hits that failed to demonstrate characteristic type II spectra (Figure 2A). The hits that showed concentration dependence with no overlapped spectra received a score of 1, while hits with any 2 or 3 overlapped spectra received scores of 2 and 3, respectively (Figure 2B–D). Compounds whose three highest concentration spectra overlapped received a score of 4 (Figure 2E). Finally, hits having all four spectra overlapped, including spectra for the highest and lowest concentrations, received a score of 5 in this system (Figure 2F).
Low throughput UV-vis binding assay
Hit validation in the low throughput UV-vis binding assay was performed according to a previously published but somewhat modified protocol [14]. UV-vis absorption spectra were recorded on a Cary scanning spectrophotometer (Varian) in the 1-cm path length quartz cuvette at 23°C in 100 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol, with CYP51 concentration of 0.5 µM. Compound solutions used for titration were prepared in DMSO at 100 µM and added in 2-µl aliquots to each 3 ml sample using a P2 pipetman (Gilson). Binding affinity of hits was estimated using the quadratic tight-binding equation [27]:(1)
where Aobs is the absorption shift determined at any ligand concentration; Amax is the maximal absorption shift obtained at saturation; KD is dissociation constant for the inhibitor-enzyme complex; S is the ligand concentration; Et is the total enzyme concentration.
Mammalian cell culture and T. cruzi maintenance
Bovine embryo skeletal muscle cells [28] or mouse C2C12 myoblasts (ATCC#CRL-1772) used in the cross-validation assay, were cultivated respectively in RPMI 1640 medium supplemented with 5% horse serum and Dulbecco's Modified Eagle's Medium H-21 containing 4.5 g/l glucose (DMEM H-21) (Cell Culture Facility, UCSF), supplemented with 5% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, MO, USA), 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin. T. cruzi CAI-72 trypomastigotes [28] were obtained from supernatant of infected cultures 4 to 7 days post-infection. Cultures were maintained at 37°C in an atmosphere of 5% CO2. To harvest parasites, the supernatant of infected cultures containing released trypomastigotes was collected and centrifuged at 600 rpm for 5 min to remove cell debris; the pellet was then discarded and the trypomastigotes remaining in the media were concentrated by spinning the culture tubes at 3300 rpm for 15 minutes. The parasite pellet was re-suspended in 2–5 ml of fresh media. Trypomastigote concentration was determined using a Neubauer hemocytometer.
Hit cross-validation in T. cruzi-infected mammalian cells
Compound cross-validation in T. cruzi infected skeletal myoblasts (bovine embryo skeletal muscle or C2C12 lineage) was performed according to the protocol adapted from Engel and co-authors [29]. Compounds stored as 10 mM stocks in DMSO were freshly diluted immediately prior to use. Final concentrations of DMSO never exceeded 0.4%, which has been shown to be non-toxic both for parasites and mammalian cells. To prepare plates for the assay, skeletal myoblasts cultured in 75–150 cm3 flasks were washed once with phosphate buffered saline (PBS) and then incubated with 0.25% trypsin in PBS for 1–3 minutes for detachment. The trypsin solution was removed and myoblasts were released from the flask bottom. Fresh medium was added and cell concentration was determined using a Neubauer hemocytometer. Myoblasts in 50 µl of culture medium were transferred to the clear-bottom wells of black 96-well plates (Greiner Bio-One, #655090) at a density of 1500 cells/well and infected with 50 µl trypomastigote culture at 7500 parasites/well (1∶5 ratio). The cells were allowed to adhere and spread for 24 h in the incubator before the media was removed and 100 µl of fresh medium containing 0, 0.04, 0.12, 0.37, 1.10, 3.30 or 10 µM of test compound was added to each well, followed by 72 h incubation at 37°C in an atmosphere of 5% CO2. Posaconazole was used as a positive control at the same concentrations as the test compounds. The cells were then fixed by adding 100 µl/well of 8% formaldehyde in PBS, resulting in a 4% working concentration of formaldehyde. To stain the nucleus of the host cell and the nucleus and kinetoplast of parasites, cell membranes were first permeabilized by washing once with 100 µl of 0.1% Triton X-100 in PBS, then treated for 1 h with 0.2 µg/ml of the fluorescent DNA dye, 4,6-diamidino-2-phenylindole (DAPI), followed by one wash with PBS. The fixed plates were kept at 4°C and protected from light until images were acquired.
Image acquisition and data analysis
Plates were allowed to equilibrate to room temperature and images were acquired by the IN Cell Analyzer 2000 (GE Healthcare) with a 20× objective, 5 fields per well, with 350/50 nm and 460/40 nm fluorescent excitation and emission filters. In the absence of drug cytotoxicity, at least 800 cells were imaged and counted per well. The images were processed and analyzed by IN Cell Analyzer Developer 1.6 software. The size parameters used to segment host and parasite organelles were 125 µm2 for host nucleus, and 1–2 µm2 for parasite nucleus/kinetoplast. Since DAPI also strongly stains host heterochromatin regions that display as shapes resembling parasites (Figure 3), particles co-localized to host nuclei were excluded from quantification, while parasite aggregates near the host nucleus were counted as infected cells. Numbers of host cells and intracellular amastigotes were determined based on host cell and parasite nucleus and parasite kinetoplast quantification, providing a measure of growth inhibition during the first 72 h of post-infection treatment compared to untreated controls.
Segmented images of DAPI-stained T.cruzi-infected myoblasts processed with image processing software are shown for cells cultured after 72 h treatment with Compounds 1 (C, E, G) or 13 (D, F, H) at indicated concentrations. Nuclei of host cells are highlighted in blue and nuclei and kinetoplasts of parasites are highlighted in red. The inserts show original DAPI staining in blue. Note the elimination of intracellular amastigotes at 0.123 µM and 10 µM of Compound 1 (E, G) and 10 µM of Compound 13 (H). In the negative control, DMSO-treated myoblasts show abundant parasitemia (A). Posaconazole was used as a positive control (B). Bars = 20 µM.
Trypanocidal assay
Compounds cross-validated with low EC50 against T. cruzi infected cells were further evaluated to determine the "cidal" concentration at which host cells are effectively cured, using the protocol adapted from Engel et al 1998 [30]. To allow long term treatment and avoid culture overgrowth, C2C12 myoblasts were seeded at low density in 96 well plates (103 cells/well) and cultured with DMEM H-21 medium supplemented with low FBS concentration (1%), 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were infected with T. cruzi trypomastigote strain CAI/72 (5 parasites/cell) for 24 h and treated with 0, 0.04, 0.12, 0.37, 1.10, 3.30 or 10 µM test compounds in 100 µl of medium for 14 days. During the course of treatment, medium supplemented with test compounds was replaced every 72 h. After 14 days, drug pressure was withdrawn and incubation continued in the compound-free medium for another week, during which cultures were monitored for reappearance of T. cruzi trypomastigotes in the supernatant.
Lipid analysis of T. cruzi amastigotes
To grow intracellular amastigotes for lipid analysis, C2C12 mouse skeletal myoblasts (4×106 cells) cultured in 150 cm2 flasks as described above were infected with 80×106 trypomastigotes for 24 h. The cultures were treated with test compounds 72 h post-infection at concentrations 5× below cidal to avoid completely killing the parasites. Posaconazole at 100 nM was used as a positive control. The cysteine protease cruzain inhibitor K777 [31] at 1.6 µM was used as a negative control to ensure that T. cruzi inhibition via other pathways did not affect composition of the membrane sterols. Cells were harvested 96 h post-infection and after 24 h of drug treatment. Cell detachment was initiated by brief treatment with 10 ml of 0.25% trypsin in PBS, followed promptly by replacement with 10 ml of fresh media containing FBS. Harvested cells were washed 3× with 25 ml of PBS to remove FBS and released trypomastigotes. The suspension of infected cells was then transferred to a glass tube and centrifuged at 2000 rpm for 5 min. After washing with PBS, 2 ml of chloroform-methanol solution (2∶1 ratio) was added to re-suspend the cell pellet. Organic solvents were then evaporated under N2 flow, allowing the pellet to dry.
For total lipid extraction, the dry cell pellet was treated with 2 ml chloroform for 24 h. Polar molecules were removed by washing the organic phase 3× with 3 ml water. The organic phase containing the sterols was subsequently dried on an evaporator under N2 flow. The lipids were re-suspended in 2 ml of chloroform-methanol (9∶1 ratio), washed again 3× with 3 ml water, dried under N2 flow and re-suspended in acetonitrile. After an additional wash 3× with 3 ml water, the acetonitrile was dried under an N2 stream. Extracted dry sterols were re-dissolved in hexane and derivatized with N,N-bis(trimethylsilyl)-2,2,2-trifluoroacetamide (BSTFA) (Pierce) by incubating at 37°C for at least 2 hours. Sterols as their trimethylsilyl (TMS) derivatives were analyzed by gas chromatography and mass spectrometry (GC-MS). GC-MS analysis and lipid identification were performed as described previously [14].
Primary screening in two-wavelength mode
Given the large size of the library, we reasoned that use of the two-wavelength detection mode would increase the throughput potential of the assay above that of the multi-wavelength (spectral) mode previously described [21], [22]. Thus, in the primary screen, detection was carried out at 425 nm and 390 nm, corresponding to the maxima and minima of type II difference binding spectra, the difference in absorbance ΔA = A425−A390 being the discriminative criterion (Figure 1). The ΔA of the DMSO control averaged 0.000±0.003 (mean ± standard deviation), while ΔA of the reference was 0.043±0.004, resulting in the assay Z′-factor value of 0.58±0.04. Standard deviation was calculated for 528 measurements. The amplitude of the absorbance difference is directly proportional to the fraction of CYP51 target bound to the compound, with the maximal value of ∼0.04 corresponding to full displacement of the iron axial water ligand as a result of saturation of the active sites. 374 compounds (0.35% of the whole library) were outliers with ΔA>0.04. We speculate that these compounds fell into the outlier category because of spectral or solubility characteristics. Twenty of these outliers were selected for re-evaluation in spectral mode and were confirmed to lack characteristic absorbance features.
Re-evaluation of two-wavelength hits in spectral screening mode
We identified false-positive hits in the two-wavelength pool by re-evaluating select compounds in spectral mode. 2,718 compounds with 0.016<ΔA<0.04 were first analyzed using Accelrys software (Accelrys, Inc., San Diego, California) for structural differences and similarities. Briefly, the fingerprint of each compound was determined using a proprietary extended-connectivity algorithm, EFCP_6, and folded to 256 bits for efficiency of comparison [32], [33]. A maximal dissimilarity [34] partitioning algorithm [35] employed the Tanimoto distance between these fingerprints to cluster the compounds into 30 groups. Similarly, 374 outliers with ΔA>0.04 were divided in 10 groups. Two representatives (one with maximum ΔA and another with median ΔA) were selected from each group, resulting in 80 compounds subjected to evaluation in full spectral mode. Evaluation was performed for four serial dilutions from 2 µM to 12 µM, followed by ranking of hits using the 0–5 scoring system (Figure 2). All 20 compounds selected from the outlier pool fell into the false-positive category (score 0). Among the remaining 60 compounds, 22 (36%) were validated as positive hits, including 7 compounds scored 4 or 5, with only one having all four spectra overlapped (score 5) (Table S2).
Selection and re-evaluation of the 475 additional two-wavelength hits in spectral mode
A visual inspection of the first 22 hits shown to be positive in both two-wavelength and spectral modes confirmed that 17 possessed nitrogen-containing aromatic heterocycles capable of coordinating to the heme iron in the active site of CYP51 (Table S2). The remaining five contained aliphatic amine groups. Informatics analysis of the entire 104,000 compound library identified 6,643 compounds having heterocycle pharmacophores with an accessible nitrogen atom in their structures; 1,274 of these were identified in the two-wavelength primary screen as demonstrating absorbance differences in the range of positive hits. Strict enforcement of the Lipinski "rule of five" [36] narrowed the pool to 1,172 compounds. Hit scaffolds, defined as consisting of >2 rings and >3 members, were clustered by structural similarity and organized into a hierarchical tree using SARvision Plus 3.1 (Altoris, San Diego) software. Based on these criteria, 188 scaffold groups plus 155 singletons were identified. 320 compounds were picked from the scaffold groups and combined with the 155 singletons, yielding 475 compounds to be re-tested in the spectral mode and ranked using the 0–5 scoring system shown in Figure 2. Among these, only 21 compounds (4%) failed to demonstrate characteristic type II spectra, while the remaining 454 compounds showed the spectral characteristics of positive hits, including 134 hits which received a score of 4, and 44 hits with a score of 5, both values suggesting low nanomolar binding affinity as approximated by equation (1). Combined with 7 previously identified hits, they constituted the 185 high affinity hits listed in the Table S3. The high target concentration dictated by the assay's sensitivity did not allow further discrimination of positive hits by their affinities in HTS format.
Based on the structure of the putative heme Fe-coordinating modules, 185 hits can be divided in six unevenly populated categories (Figure 4A). The number of compounds populating each category varied from 1 to 92. This distribution reflects the frequency of each category in the library (Figure 4B), suggesting a lack of preference for the Fe-coordinating heterocycle as scored by this assay. Each of the 6 categories was further subdivided into clusters sharing more extensive structural similarities among the members (Figure S1). Some clusters had a higher number of T. cruzi-active hits (highlighted green in Figure S1) than others.
Distribution of nitrogen-containing aromatic heterocyclic pharmacophores among 185 positive hits with binding score 4 or 5 (A) echoes their frequency in the library (B). R represents diverse chemical structures as shown in Table S3.
Cross-validation of hits in T. cruzi-infected mammalian cells
The set of 475 compounds plus the 80 hits previously validated in spectral mode were cross-validated in T. cruzi-infected myoblast cells using an image-based medium throughput assay [29] (Figure 3). Posaconazole was used as a positive control with an EC50 of 1 nM. EC50 was determined as a concentration of compound corresponding to 50% inhibition of parasite endocytosis and intracellular growth, the calculation being based on the sigmoidal dose-response curve of compound-induced inhibition of the T. cruzi infection index (i.e., the percentage of T. cruzi-infected cells in culture multiplied by the mean number of parasites per infected cell).
Of the 497 cross-validated compounds, 57 exhibited anti-T. cruzi activity with EC50 below 10 µM; this group was notably enriched with CYP51 binding hit scores of 4 and 5 (36 of 57 total hits) (Table 1). The remaining hits (21 of 57 total hits, including the highest rated hit) had binding scores ranging from 1 to 3. The complete list of the 57 T. cruzi-active hits is shown in Table S4. A subset of eleven sub-micromolar hits is shown in Figure 5. Numbering of compounds in Figure 5 and Table S4 is according to their descending rank order. Both the 185-compound spectral pool (http://zinc.docking.org/users/lpodust/carts/185CYP51spectralHTShits) and the 57-compound T. cruzi-active pool (http://zinc.docking.org/users/lpodust/carts/57Tcruziactivehits) were uploaded to and made publicly available through the free online resource ZINC (http://zinc.docking.org) [37].
aEC50 values obtained against T. cruzi parasites in HTS assay used to rank order the hits. bIn parenthesis are EC50 averaged from two independent cross-validation assays for individually repurchased hits.
Comparative analysis of both spectral and T. cruzi-active pools by logP and MW distributions showed good correlation between the two pools (Figure 6). A slight enrichment of hits with logP >3 in the T. cruzi active pool (82.5% in the T. cruzi-active pool vs. 78.9% in the pool of spectral hits) was observed (Figure 6A), with the molecular weight being rather evenly distributed across the range of 200–400 g/mol, with only a few hits crossing the 400 g/mol barrier (Figure 6B). Compound 5 with MW of 225 and calculated logP of 2.6 was the smallest molecule ranked among the top five T. cruzi-active hits.
Distribution of the (A) logP and (B) molecular weight (MW) values.
Compound 1, [6-(2-bromophenoxy)hexyl]imidazole, (MW of 323, logP of 4.2) was ranked first with an EC50 of 70 nM and HTS binding score of 2. Upon more rigorous validation in low throughput assays, Compound 1 was shown to have at least low nanomolar binding affinity to the target (Figure 7) and an EC50 of 17±12 nM averaged from the two independent assays against T. cruzi amastigotes cultured in mouse myoblasts (Figure 3). Trypanocidal concentration of Compound 1 was 40 nM in three independent experiments, which compares favorably to posaconazole (EC50 of 1 nM, trypanocidal at 0.6 nM).
(A) Hit compound structures. Compound 13 shows structural similarity to miconazole. Stoichiometric binding of 1 (B) and 13 (C) to CYP51 at 0.5 µM by UV-vis titration suggests low nanomolar binding affinity as approximated by quadratic tight-binding equation (1).
Re-mining data for sub-micromolar T. cruzi hits
Re-mining spectral screening data using the highly rated hits as molecular scaffolds, emulated a mini-SAR series providing insight to structure-activity relationships of the hits. For instance, Compound 1 was structurally distinct compared to azole antifungal drugs and the majority of positive hits identified in these studies. Two other highly ranked compounds, 4 and 11, resembled Compound 1 (Figure 5). Altogether, seven structurally related hits were characterized by binding scores of 4 or 5, and two hits, including Compound 1, by scores of 2 (Figure 8). In addition, two commercial analogs which were not part of the screened library, Compounds 1a and 1b, were tested. Compound 1a differs by the position of the bromine atom in the bromophenoxy ring, whereas 1b by the length of the alkyl linker between imidazole and bromophenoxy rings, both parameters seem essential for anti-T. cruzi activity (Figure 8). All variations presented in Figure 8 are associated with reduced EC50, either due to drop in binding affinity to the target (e.g., Compound 1b or Compound 11), or possibly to other factors. Thus, relocation of the bromine atom from the meta- to the para-position of the bromophenoxy ring (Compound 1a) results in an order of magnitude drop of anti-T. cruzi activity without detectable change in KD. However, an enzyme concentration of 0.5 µM defines the lower limit of the method's sensitivity at a KD of ∼5 nM, a hundredth of the target concentration, if a plateau in the titration curve is reached at the stoichiometric enzyme-ligand ratio (Figure 7 B, C). As differences in affinity between very tight-binding ligands are reflected in the sharpness of the titration curve, KD values recovered from fits to experimental data approximated by equation (1) are disproportionately sensitive to error in data points near the inflection point. Therefore, caution should be exercised when comparing the tight binding constants.
aEC50 values obtained against T. cruzi parasites in HTS assay used to rank order the hits. bIn parenthesis are EC50 averaged from two independent cross-validation assays for individually repurchased hits. N/A – not applicable; N/D – not determined.
Compounds 2, 6 and 7, highly rated in cross-validation, belong to their own molecular scaffold cluster, which includes 21 hits (Table S5). Hits rated highest in spectral mode (score 4 or 5) or in T. cruzi assay are summarized in (Figure 9). Compound 6, methyl 2-amino-6-[2-(3,4-dimethoxyphenyl)ethyl]-7-methyl-5-oxo-4-pyridin-3-yl-5,6-dihydro-4H-pyrano[3,2-c]pyridine-3-carboxylate, was the only one from this cluster validated in low throughput format, including GC-MS analysis of sterol composition in treated amastigotes (not shown). Based on this validation, Compound 6 is the runner-up in the series of individually tested compounds, with estimated KD of 76 nM and EC50 of 80 nM. Further validation of Compound 2 and other representatives of this cluster are pending commercial availability of the compounds.
aEC50 values obtained against T.cruzi parasites in HTS assay used to rank order the hits. bIn parenthesis is the EC50 obtained in validation assays for individually repurchased hits. N/A - not applicable; N/D - not determined.
Hit selection for manual validation
Two criteria – anti-T. cruzi activity and/or high screening score – were used to select commercially available compounds for more rigorous validation, including impact on the sterol biosynthesis in T. cruzi amastigotes cultured in mouse myoblasts. A search for compound vendors was conducted using the online resource ZINC [37]. Altogether, 32 hit compounds were acquired for validation in manual low-throughput assays, including eight T. cruzi-active hits: Compounds 1, 3, 5, 6, 9, 11, 13 and 15 (Table S6). Binding could not be confirmed for two of the acquired compounds due to compound precipitation under assay conditions. The other repurchased compounds bound CYP51 with one-to-one stoichiometry at 0.5 µM protein concentration (Figure 7), suggesting at least low nanomolar binding affinity as extrapolated from equation (1). Low EC50 values were confirmed for all tested T. cruzi–active hits (Figure 5).
GC-MS analysis of membrane sterols
Inhibition of sterol biosynthesis by GC-MS analysis was confirmed for all individually acquired hits. The major sterol observed in untreated amastigotes was episterol (peak d in Figure 10), followed by approximately equal amounts of fecosterol (e) and cholesta-7,24-dien-3β-ol (peak a). This sterol composition is consistent with our previously published work [14] but differs from Liendo and co-authors [5], where 24-methyl-7-en-cholesta-en-3β-ol (peak c in Figure 10) was identified as a main sterol of intracellular amastigotes. The most straightforward explanation for these differences would be the differences in the lipid extraction protocols used in this work or published elsewhere [38]. As a result of CYP51 inhibition, two 14-methylated precursors, lanosterol (f) and eburicol (h) dominated the GC-MS traces of the strongest CYP51 inhibitors posaconazole and Compound 1, and were present in amounts comparable to those of 14-demethylated products for the less potent Compound 13 (Figure 10). Interestingly, fecosterol (e) was completely inhibited by low concentrations of posaconazole and Compound 1, whereas episterol (d) persisted. This observation seems difficult to explain in light of the T. cruzi sterol pathway reported elsewhere [5], in which fecosterol precedes episterol in the reaction chain.
DMSO and K777 were used as negative controls; posaconazole served as a positive control. Uninfected host cell panel (top) demonstrates that chromatographic peaks labeled with small-case letters from a to i are of T. cruzi origin, corresponding to a - cholesta-7,24-dien-3β-ol, [M]•+ = m/z 454; b - cholesta-8,24-dien-3β-ol (zymosterol), [M]•+ = m/z 470; c - 24-methyl-7-en-cholesta-en-3β-ol, [M]•+ = m/z 472; d - ergosta-7,24-diene-3β-ol (episterol), [M]•+ = m/z 470; e - ergosta-8,24-diene-3β-ol (fecosterol), [M]•+ = m/z 470; f - lanosterol, [M]•+ = m/z 498; g - 4-methylepisterol, [M]•+ = m/z 484; h - eburicol, [M]•+ = m/z 512; i - 24-ethyl-7,24(24′)-en-cholesta-dien-3β-ol, [M]•+ = m/z 484. Cholesterol is the only peak originating from host cells. Increase in the CYP51 substrates lanosterol (f) and eburicol (h) is due to inhibition of T. cruzi CYP51, as is the decline in the downstream major products episterol (d), fecosterol (e), cholesta-7,24-dien-3β-ol (a) and the minor products 4-methyl-episterol (g) and 24-ethyl-7,24(24′)en-cholestadien-3β-ol (i).
Cheminformatic analysis of hits
Given an unexpectedly large number of structurally diverse hits, we used an algorithm implemented in the online cheminformatic research tool SEA (Similarity Ensemble Approach) (http://sea.bkslab.org/) [39] to relate CYP51 to other pharmacologic targets by ligand topology. In SEA, the similarity of protein targets is established and scored based on the similarity of their ligands using the probability of a random match, or expectation E-value, as a scoring function that relates lower E-values to more statistically significant ligand similarities. Results from probing the molecule databases implemented in SEA [39] with the 57 T. cruzi-active hits, either individually or as a pool, indicated CYP51's similarity to other P450 drug targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17), and aromatase (CYP19). These enzymes have been targeted by the pharmaceutical industry for cardiovascular disease [40], metabolic disorders of lipid metabolism and inflammation [41], prostate cancer [42], and estrogen receptor-positive breast cancer [43], respectively. Unexpectedly, these pharmacologic targets scored substantially higher than sterol 14α-demethylase (CYP51) itself, giving credence to the idea that molecules directed against these targets, including drugs that have entered the clinic, could be examined for potency against T. cruzi CYP51.
Another surprise revealed by cheminformatic searching in the SEA databases is the striking resemblance of the highest rated hit to inhibitors of glutaminyl-peptide cyclotransferase, an enzyme unrelated to CYP family either by sequence or structure. This enzyme catalyzes the formation of N-terminal pyroglutamic acid whose biological function is either to mediate peptide hormone interaction with receptors, or to stabilize proteins and peptides against N-terminal degradation [44]. Human glutaminyl-cyclase which catalyzes pyroglutamic acid formation at the N-terminus of amyloid peptides, is potentially involved in the development and progression of neurodegenerative disorders [44].
We designed an HTS assay to identify compounds capable of coordinating to the heme iron of CYP51. Following target-based screening and the further validation steps summarized in Figure 11, we identified 185 high affinity hits and 57 partially overlapping hits with anti-T. cruzi activity, including a dozen sub-micromolar inhibitors of T. cruzi infection in mammalian cells. Hits identified in the primary two-wavelength screen were evaluated by cheminformatic analysis, spectral screening and cross-validation against T. cruzi amastigotes. Informatics feedback served to maximize scaffold diversity and reduce the number of compounds subject to re-evaluation in the more laborious spectral mode. As a result, the two pools, of 185 and 57 hits, were structurally diverse with respect to the number, size and arrangement of rings, as well as the nature of the linkers and substituting groups constituting the compound frameworks (Tables S3 and S4). In summary, although two-wavelength mode is more efficient in terms of throughput, it generated >60% false-positive hits. Although more time-consuming to read, analyze and tabulate, the spectral mode is a more comprehensive methodology than the two-wavelength mode in terms of efficiently filtering and ranking true positive hits. Overall, based on validation tests for representative compounds, we expect the set of hits identified in this work to contain a high percentage of true positive hits and to be a valuable resource for further development of Chagas Disease applications.
At the high hit rate we observed, we were unlikely to miss important chemotypes by excluding 102 compounds violating Lipinski rule of five from validation in the full spectral mode and cross-validation in the T. cruzi assay. The process of optimization generally yields larger and more hydrophobic compounds, and thus it is industry practice to focus on smaller "lead-like" compounds to allow MW and logP to increase during lead development [45]. On the other hand, a rationale for looking at hydrophobic hits in more detail is justified by the fact that the CYP51 inhibitors itraconazole and posaconazole do not comply with this rule. The potency of posaconazole in the treatment of invasive fungal infections is attributed to accumulation of drugs in the tissues of internal solid organs rather than to free levels of drug in plasma [46], [47]. In isolated cells and solid tissues, both posaconazole and itraconazole are mostly concentrated in cellular membranes, which are also the site of their molecular target, CYP51 [48]. Given that the potent in vivo activity of posaconazole and other novel CYP51 inhibitors is attributed to their special pharmacokinetic properties, such as large volumes of distribution and long terminal half-life [49], [50], it may be worth revisiting the 102 hits initially excluded by the Lipinski criteria.
The T. cruzi–active pool contained both high affinity hits (score 4 or 5) and those with lower binding scores (Table 1), suggesting that low score compounds might have been acting via alternative mechanisms. To test this possibility, the binding and activity of the highest rated hit, Compound 1, was confirmed in manual assays. Compound 1, had activity an order of magnitude higher than any other hit identified in these studies (Figure 5). At 100 nM concentration, Compound 1 suppressed sterol biosynthesis in T. cruzi amastigotes more efficiently than posaconazole (Figure 10), thus becoming a candidate for further evaluation in an animal model of infection. Cheminformatic searching in the SEA databases [39] revealed a significant resemblance of the top hit to inhibitors of P450 drug targets involved in eicosanoid synthesis and fatty acid metabolism. For example, thromboxane-A synthase was highly ranked with regard to the top hit and the structurally related sub-micromolar hits 4 and 11, as were the fatty acid ω-hydroxylases of the CYP4 family. Surprisingly, the lowest E-values were obtained for glutaminyl-peptide cyclotransferase, which is unrelated to CYP family by sequence or structure. While the ability of drugs to bind to unintended targets in the human host can lead to undesirable side effects, the same binding to a target in a pathogen infecting a human can cure infection. In the context of a deficit of drugs for neglected diseases, the unexpected promiscuity of CYP51 encountered in this work could be utilized in a "piggy-back" strategy [51], [52] for identifying a chemical starting point for anti-parasitic therapy development, while structure-activity relationships derived from parasite assays could lead to disease-specific clinical candidates [53]–[56].
The structural motif present in the cohort of antifungal azole drugs was apparent in both pools. For instance, hit 1, 2-(4-chlorophenoxy)-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone (binding score 5) (Compound 13), which ranked thirteenth in the anti-T. cruzi activity assay, resembled miconazole (Figure 7A), one of the first broad-spectrum antimycotic agents and the conazole progenitor of orally active antifungal agents such as ketoconazole, itraconazole, posaconazole, fluconazole and voriconazole [15]. Manual UV-vis titration confirmed that Compound 13 binds and saturates CYP51 at equimolar 0.5 µM concentrations, suggesting at least low nanomolar binding affinity (Figure 7C). According to the SciFinder online resource, compounds with close structural similarities to Compound 13 were patented in the 1970's as potent fungicides. To confirm a relationship between this structural motif and biological activity, we extracted membrane sterols from T. cruzi amastigotes isolated from infected mouse myoblasts treated with Compound 13. Consistent with the inhibition of CYP51, GC-MS analysis showed an increase in lanosterol and eburicol precursors and a concomitant decline in episterol, fecosterol and other 14-demethylated intermediates (Figure 10). Compound 13 was effective against T. cruzi amastigotes with an EC50 of 1.3 µM and was trypanocidal at 2.5 µM.
The shape of the posaconazole molecule defines its binding mode to CYP51. Its long "tail" group extends into the substrate-binding tunnel [24], [57]. In the mouth of the tunnel, posaconazole adopts alternative conformations and makes multiple points of contact with amino acid residues on the protein surface [24]. Extensive interactions of posaconazole with catalytically non-essential residues in CYP51 are strikingly consistent with the pattern of drug resistance in fungi. The points of posaconazole contact are mutation hot spots in azole-resistant isolates of the pathogenic fungi Aspergillus fumigatus and Candida albicans [24]. Some of these mutations confer cross-resistance to all azole drugs [58]–[61]. The spread of drug resistance due to mutation of the target in response to therapy might be avoided in T. cruzi if the binding properties of new inhibitors were improved using target structure as a guide. This concept has been successfully used for a panel of 9 FDA-approved HIV protease inhibitors developed with extensive use of structure-based drug design [62]. In order to stay within a substrate envelope and focus hit-to-lead optimization chemistry on main-chain interactions, we are pursuing co-crystallization of low molecular weight hits with CYP51 for x-ray structure analysis. Compared to the larger posaconazole (MW 700 g/mol), the molecular weight of hits active against T. cruzi ranged from 215 to 480, with the majority (39 of 57) below 360; Compounds 1 and 13 are 323 and 348 g/mol, respectively. Furthermore, with its "fragment-like" characteristics [63] of the lowest molecular weight and logP among the sub-micromolar T. cruzi hits, Compound 5 has good prospects in hit-to-lead optimization, as does one of the smallest high affinity spectral hits, 5,8-dibromoisoquinoline (Compound 45 in Table S3; MW 287 g/mol), consisting only of an iron-coordinating heterocyclic module.
In conclusion, diversification of leads for CYP51 inhibitors should offer the medicinal chemist new choices in terms of chemical accessibility and prospects for lead optimization. Multiple leads lower the risk of drug attrition in the case of undesirable ADMET properties and also cover a patent-free space, one of the keys to the successful anti-Chagasic therapy of the future. In the course of this work we have (i) further developed a UV-based high throughput screening methodology for P450 enzymes; (ii) diversified the chemical scaffold space for CYP51 inhibitors; (iii) identified a potent T. cruzi inhibitor and a diverse array of low molecular weight hits with high affinity to CYP51 for hit-to-lead optimization; (iv) related CYP51 to other pharmacologic targets by computational ligand chemistry. This effort has allowed us to identify molecules already produced by pharmaceutical companies for future experimental testing against T. cruzi.
Supporting Information
Figure S1.
Clustering of the 185 hits validated with the binding score 4 or 5.
Table S1.
Analysis of plate readings.
Table S2.
First 22 hits validated positive both in two-wavelength and spectral screening modes.
Table S3.
185 hits validated with binding score 4 or 5.
Table S4.
57 T. cruzi -active hits in descending rank order.
Table S5.
Second highest rated hit compound cluster.
Table S6.
32 individually validated hits.
Alternative Language Abstract S1.
Manuscript Abstract in Portuguese.
We thank John Irwin for valuable discussions and Potter Wickware for critical reading of the manuscript.
Author Contributions
Conceived and designed the experiments: JHM MRA LMP. Performed the experiments: SSG CMC JBJ CKC JG JCE KKHA. Analyzed the data: SSG CMC JBJ CKC GE KKHA JM SC LMP. Contributed reagents/materials/analysis tools: JBJ GE JCE JM SC MRA JHM LMP. Wrote the paper: SSG CMC LMP.
- 1. Chagas C (1909) Nova trypanozomiaze humana: estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n. sp., ajente etiolojico de nova entidade morbida do homem. Mem Inst Oswaldo Cruz 1: 159–218.
- 2. Maya JD, Cassels BK, Iturriaga-Vasquez P, Ferreira J, Faundez M, et al. (2007) Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host. Comp Biochem Physiol, Part A Mol Integr Physiol 146: 601–620.
- 3. Rassi A Jr, Rassi A, Marin-Neto JA (2010) Chagas disease. Lancet 375: 1388–1402.
- 4. Buckner FS, Navabi N (2010) Advances in Chagas disease drug development: 2009–2010. Curr Opin Infect Dis 23: 609–616.
- 5. Liendo A, Visbal G, Piras MM, Piras R, Urbina JA (1999) Sterol composition and biosynthesis in Trypanosoma cruzi amastigotes. Mol Biochem Parasitol 104: 81–91.
- 6. Roberts CW, McLeod R, Rice DW, Ginger M, Chance ML, et al. (2003) Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol Biochem Parasitol 126: 129–142.
- 7. de Souza W, Rodrigues JC (2009) Sterol biosynthesis pathway as target for anti-trypanosomatid drugs. Interdiscip Perspect Infect Dis 2009: 642502.
- 8.
Buckner F (2008) Sterol 14-demethylase inhibitors for Trypanosoma cruzi infections. In: Drug Targets in Kinetoplastid Parasites. Majumder HK, editor. New York: Springer. 61–80.
- 9. Ferraz ML, Gazzinelli RT, Alves RO, Urbina JA, Romanha AJ (2007) The anti-Trypanosoma cruzi activity of posaconazole in a murine model of acute Chagas' disease is less dependent on gamma interferon than that of benznidazole. Antimicrob Agents Chemother 51: 1359–1364.
- 10. Urbina JA (2009) Ergosterol biosynthesis and drug development for Chagas disease. Mem Inst Oswaldo Cruz 104 Suppl 1: 311–318.
- 11. Olivieri BP, Molina JT, de Castro SL, Pereira MC, Calvet CM, et al. (2010) A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease. Int J Antimicrob Agents 36: 79–83.
- 12. Pinazo M-J, Espinosa G, Gállego M, López-Chejade PL, Urbina J, et al. (2010) Treatment with posaconazole of a patient with systemic lupus erythematosus and Chagas Disease. Am J Trop Med Hyg 82: 583–587.
- 13. Lazardi K, Urbina JA, de Souza W (1990) Ultrastructural alterations induced by two ergosterol biosynthesis inhibitors, ketoconazole and terbinafine, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob Agents Chemother 34: 2097–2105.
- 14. Doyle PS, Chen C-K, Johnston JB, Hopkins SD, Leung SSF, et al. (2010) A nonazole CYP51 inhibitor cures Chagas' Disease in a mouse model of acute infection. Antimicrob Agents Chemother 54: 2480–2488.
- 15. Heeres J, Meerpoel L, Lewi P (2010) Conazoles. Molecules 15: 4129–4188.
- 16. Kauffman CA, Malani AN, Easley C, Kirkpatrick P (2007) Posaconazole. Nat Rev Drug Discov 6: 183–184.
- 17. Katragkou A, Tsikopoulou F, Roilides E, Zaoutis TE (2011) Posaconazole: when and how? The clinician's view. Mycoses 55: 110–122.
- 18. Urbina JA, Payares G, Molina J, Sanoja C, Liendo A, et al. (1996) Cure of short- and long-term experimental Chagas' disease using D0870. Science 273: 969–971.
- 19. Urbina JA, Payares G, Contreras LM, Liendo A, Sanoja C, et al. (1998) Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies. Antimicrob Agents Chemother 42: 1771–1777.
- 20. Schenkman JB, Remmer H, Estabrook RW (1967) Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol Pharmacol 3: 113–123.
- 21. von Kries JP, Warrier T, Podust LM (2010) Identification of small-molecule scaffolds for P450 inhibitors. Curr Protoc Microbiol 16: Unit 17.14.
- 22. Podust LM, von Kries JP, Nasser Eddine A, Kim Y, Yermalitskaya LV, et al. (2007) Small molecule scaffolds for CYP51 inhibitors identified by high-throughput screening and defined by x-ray crystallography. Antimicrob Agents Chemother 51: 3915–3923.
- 23. Chen C-K, Doyle PS, Yermalitskaya LV, Mackey ZB, Ang KKH, et al. (2009) Trypanosoma cruzi CYP51 inhibitor derived from a Mycobacterium tuberculosis screen hit. PLoS Negl Trop Dis 3: e372.
- 24. Chen C-K, Leung SSF, Guilbert C, Jacobson MP, McKerrow JH, et al. (2010) Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole. PLoS Negl Trop Dis 4: e651.
- 25. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J Biol Chem 239: 2379–2385.
- 26. Ouellet H, Kells PM, Ortiz de Montellano PR, Podust LM (2010) Reverse type I inhibitor of Mycobacterium tuberculosis CYP125A1. Bioorg Med Chem Lett 21: 332–337.
- 27. Morrison JF (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta 185: 269–286.
- 28. Engel JC, Doyle PS, Dvorak JA (1985) Trypanosoma cruzi: biological characterization of clones derived from chronic chagasic patients. II. Quantitative analysis of the intracellular cycle. J Protozool 32: 80–83.
- 29. Engel JC, Ang KKH, Chen S, Arkin MR, McKerrow JH, et al. (2010) Image-based high-throughput drug screening targeting the intracellular stage of Trypanosoma cruzi, the agent of Chagas' Disease. Antimicrob Agents Chemother 54: 3326–3334.
- 30. Engel JC, Doyle PS, Hsieh I, McKerrow JH (1998) Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection. J Exp Med 188: 725–734.
- 31. McKerrow JH, Doyle PS, Engel JC, Podust LM, Robertson SA, et al. (2009) Two approaches to discovering and developing new drugs for Chagas disease. Mem Inst Oswaldo Cruz 104: 263–269.
- 32. Hassan M, Brown R, Varma-O'Brien S, Rogers D (2006) Cheminformatics analysis and learning in a data pipelining environment. Mol Divers 10: 283–299.
- 33. Rogers D, Hahn M (2010) Extended-Connectivity Fingerprints. J Chem Inf Model 50: 742–754.
- 34. Hassan M, Bielawski JP, Hempel JC, Waldman M (1996) Optimization and visualization of molecular diversity of combinatorial libraries. Mol Divers 2: 64–74.
- 35. Kaufman L, Rousseeuw P (1990) Finding Groups in Data: An Introduction to Cluster Analysis. Hoboken, New Jersey: John Wiley & Sons.
- 36. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 46: 3–26.
- 37. Irwin JJ, Shoichet BK (2005) ZINC–a free database of commercially available compounds for virtual screening. J Chem Inf Model 45: 177–182.
- 38. Urbina JA, Lazardi K, Aguirre T, Piras MM, Piras R (1991) Antiproliferative effects and mechanism of action of ICI 195,739, a novel bis-triazole derivative, on epimastigotes and amastigotes of Trypanosoma (Schizotrypanum) cruzi. Antimicrob Agents Chemother 35: 730–735.
- 39. Keiser MJ, Roth BL, Armbruster BN, Ernsberger P, Irwin JJ, et al. (2007) Relating protein pharmacology by ligand chemistry. Nat Biotechnol 25: 197–206.
- 40. Sakariassen KS, Alberts P, Fontana P, Mann J, Bounameaux H, et al. (2009) Effect of pharmaceutical interventions targeting thromboxane receptors and thromboxane synthase in cardiovascular and renal diseases. Future Cardiology 5: 479–493.
- 41. Hardwick JP (2008) Cytochrome P450 omega hydroxylase (CYP4) function in fatty acid metabolism and metabolic diseases. Biochem Pharmacol 75: 2263–2275.
- 42. Courtney KD, Taplin ME (2012) The evolving paradigm of second-line hormonal therapy options for castration-resistant prostate cancer. Curr Opin Oncol DOI:https://doi.org/10.1097/CCO.0b013e328351059d.
- 43. Litton JK, Arun BK, Brown PH, Hortobagyi GN (2012) Aromatase inhibitors and breast cancer prevention. Expert Opin Pharmaco 13: 325–331.
- 44. Schilling S, Wasternack C, Demuth HU (2008) Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution. Biol Chem 389: 983–991.
- 45. Oprea TI (2002) Current trends in lead discovery: Are we looking for the appropriate properties? J Comput-Aided Mol Des 16: 325–334.
- 46. Lignell A, Lowdin E, Cars O, Chryssanthou E, Sjolin J (2011) Posaconazole in human serum: a greater pharmacodynamic effect than predicted by the non-protein-bound serum concentration. Antimicrob Agents Chemother 55: 3099–3104.
- 47. Dolton MJ, Ray JE, Marriott D, McLachlan AJ (2012) Posaconazole exposure-response relationship: evaluating the utility of therapeutic drug monitoring. Antimicrob Agents Chemother
- 48. Campoli P, Al Abdallah Q, Robitaille R, Solis NV, Fielhaber JA, et al. (2011) Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis. Antimicrob Agents Chemother 55: 5732–5739.
- 49. Urbina JA (1999) Chemotherapy of Chagas' disease: the how and the why. J Mol Med 77: 332–338.
- 50. Urbina JA (2010) Specific chemotherapy of Chagas disease: relevance, current limitations and new approaches. Acta Trop 115: 55–68.
- 51. Nwaka S, Hudson A (2006) Innovative lead discovery strategies for tropical diseases. Nat Rev Drug Discov 5: 941–955.
- 52. Gelb MH, Van Voorhis WC, Buckner FS, Yokoyama K, Eastman R, et al. (2003) Protein farnesyl and N-myristoyl transferases: piggy-back medicinal chemistry targets for the development of antitrypanosomatid and antimalarial therapeutics. Mol Biochem Parasitol 126: 155–163.
- 53. Hucke O, Gelb MH, Verlinde CL, Buckner FS (2005) The protein farnesyltransferase inhibitor Tipifarnib as a new lead for the development of drugs against Chagas disease. J Med Chem 48: 5415–5418.
- 54. Chennamaneni NK, Arif J, Buckner FS, Gelb MH (2009) Isoquinoline-based analogs of the cancer drug clinical candidate tipifarnib as anti-Trypanosoma cruzi agents. Bioorg Med Chem Lett 19: 6582–6584.
- 55. Kraus JM, Verlinde CL, Karimi M, Lepesheva GI, Gelb MH, et al. (2009) Rational modification of a candidate cancer drug for use against Chagas disease. J Med Chem 52: 1639–1647.
- 56. Kraus JM, Tatipaka HB, McGuffin SA, Chennamaneni NK, Karimi M, et al. (2010) Second generation analogues of the cancer drug clinical candidate tipifarnib for anti-Chagas disease drug discovery. J Med Chem 53: 3887–3898.
- 57. Lepesheva GI, Hargrove TY, Anderson S, Kleshchenko Y, Furtak V, et al. (2010) Structural insights into inhibition of sterol 14alpha-demethylase in the human pathogen Trypanosoma cruzi. J Biol Chem 285: 25582–25590.
- 58. Mann PA, Parmegiani RM, Wei SQ, Mendrick CA, Li X, et al. (2003) Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14alpha-demethylase. Antimicrob Agents Chemother 47: 577–581.
- 59. Mellado E, Garcia-Effron G, Alcazar-Fuoli L, Cuenca-Estrella M, Rodriguez-Tudela JL (2004) Substitutions at methionine 220 in the 14alpha-sterol demethylase (Cyp51A) of Aspergillus fumigatus are responsible for resistance in vitro to azole antifungal drugs. Antimicrob Agents Chemother 48: 2747–2750.
- 60. Rodriguez-Tudela JL, Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Monzon A, et al. (2008) Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus. Antimicrob Agents Chemother 52: 2468–2472.
- 61. Pinto e Silva AT, Costa-de-Oliveira S, Silva-Dias A, Pina-Vaz C, Rodrigues AG (2009) Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles. FEMS Yeast Res 9: 626–633.
- 62. Ali A, Bandaranayake RM, Cai Y, King NM, Kolli M, et al. (2010) Molecular basis for drug resistance in HIV-1 protease. Viruses 2: 2509–2535.
- 63. Verdonk ML, Hartshorn MJ (2004) Structure-guided fragment screening for lead discovery. Curr Opin Drug Discov Devel 7: 404–410.


text:
Business Secretary Vince Cable indicated he is considering reforms to the "public interest test" for corporate takeovers as a political row erupted about US pharmaceutical giant Pfizer's move for AstraZeneca.
Mr Cable said the Government had been "looking at the options around the public interest test" after Labour leader Ed Miliband demanded a full assessment of the potential deal.
AstraZeneca spurned a new £63 billion takeover offer from US rival Pfizer last week, saying it "substantially" undervalued the business and the pharmaceutical giant is reported to be considering its next move.
The Labour leader has written to David Cameron calling for the publication of any analysis the Government has made of the proposal and claimed the Prime Minister had been acting as a "cheerleader" for the proposed deal.
He proposed widening the scope of the public interest test to take in industries of strategic importance - such as the high-value scientific research carried out by AstraZeneca.
"If I was prime minster I would be bringing in a new public interest test ... widening the scope of the current public interest test, which says that when we have the largest takeover in British history, with all the implications it has for our science base, there's got to be an independent assessment of the implications of this for our national interest," he told BBC1's Andrew Marr Show.
The Government can currently intervene on mergers or takeovers where there is a national security issue, an issue of media plurality, competition concerns or if it could affect financial stability.
Asked about reviewing the test, the Business Secretary said: "Obviously we are looking at this option amongst others, I'm sure you wouldn't expect me to go into all the detail and the discussions we are currently having between the two parties to the proposal.
"What we are endeavouring to ensure in the Government is to make absolutely sure Britain's excellent science base and pharmaceutical research is properly protected, manufacturing in the UK, decision-making, those are the issues we are talking to the companies about."
Mr Cable said he had spoken to the chief executives of both firms and stressed that the Government was "strictly neutral" between the two.
Mr Cameron has said the Government had received "robust" assurances from Pfizer about protection for British jobs and research under the proposed deal and stressed it was a decision for shareholders.
The Liberal Democrat Business Secretary said there was a "lot of small print to study" in the assurances given by Viagra manufacturer Pfizer.
"We publicly welcome the fact that a letter has been issued by the company which underlines the importance we attach in Government to protecting British science and jobs and investment and manufacturing and decision-making in the UK but obviously there is a lot of small print to study, the question about how these obligations are binding and of course we're talking about two companies here and we're strictly neutral as between the two."
He said that the British competition and merger regime was non-political in the way it applied "but there is an important national interest here".
Mr Cable's comments were in contrast to Tory attacks on Mr Miliband over his intervention in the row.
Conservative Party chairman Grant Shapps accused the Labour leader of "playing politics with people's jobs" and being
"anti-business in every single way".
He told Channel 4 News: "We won't cheerlead for a bid one way or the other but we will be absolutely certain if it goes ahead that it's good for jobs, good for research and development - particularly in some of those life science areas where Britain has a really strong lead - and that it benefits the country.
"Those are the simple tests and it can't go ahead unless it does all those things."
Mr Miliband said "no other country in the world would be waving this bid through" and claimed the Government had been given "pretty weak assurances" by the US firm which had a "pretty dubious record when it comes to their record in this country and other takeovers".
In his letter to Mr Cameron the Labour leader said: "I am strongly of the view that, when it comes to such a strategically important part of UK PLC, we need a more substantive assessment of whether this takeover is in the national economic interest before the UK Government allows itself to be seen to be supporting it.
"There are reasons to think that such an assessment would be of value. Firstly the company's track record in the UK raises concerns, including their decision in 2012 to close down the world-renowned R&D facility in Sandwich, leading to the loss of 1,500 jobs.
"Secondly AstraZeneca currently spends a significantly higher proportion of its turnover on R&D than Pfizer - a crucial concern given the importance of pharmaceutical investment in R&D to the UK science base.
"Lastly there are concerns about Pfizer's wider track record regarding the impact on research of previous takeovers."

subset: commoncrawl


text:
0 Members and 1 Guest are viewing this topic.
Anyone remember the good old days of CONCEPT-5?
The good thing about the OFP editing community is that you get your reputation with your achievements and not with the amount of pointless waffle you produce.
by: fragstaThats a good point. I haven't acheived anything though...yet....
Neah neah improved missions back homie? neah super cool editing skills, compared to where ya were standing before I can hardly believe my ears!
Problems with the site? Email us
Disclaimer | Privacy Policy


text:
Some have asked for more detail on the Midwest Rivendell Rally I mentioned earlier, so here's as much as I can commit to nearly 4 months in advance. The ride will be a 2-day circuit around Lake Pepin, much like the 3-speed tour. The dates are June 1-3, with the actual riding being on June 2-3. The event will formally kick-off on June 1 in Red Wing, where out-of-towners and others who choose to spend a night in Red Wing can gather for a social event that evening. I plan to ride down to Red Wing from Minneapolis on June 1. Anybody is welcome to join me on that ride. Anybody who drives to Red Wing should find a legal place to park the car for the weekend, but that should be easy enough. And of course, anybody who is spending that night in Red Wing will need to figure out lodging.
First of all, I would like to clarify what level of support I plan to provide for this, and what each participant will have to arrange for himself or herself. I will provide a route and a basic schedule. I will not provide food or lodging, but it will be easy enough for you to arrange those things yourself (see below). Hand-holding and babysitting will be supplied on an extremely limited basis, so if you're looking for a fully supported tour, this probably won't be your cup of tea. I might be able to arrange a support vehicle of some sort. This is tentative, but seems like a decent idea. The support vehicle may be for emergencies only or it may be to carry your luggage, but that will depend on the size of the vehicle (if any) and how many people need to have stuff carried. Plan to carry what you need and have your body and your bike in good operating condition.
I will provide a suggested route, which is somewhat but not insanely challenging, and an alternate "easy" route for those who feel the main route is too rugged.
On Saturday, we will ride the Wisconsin side of the lake. The main route will include some steep climbs/descents and a few "rustic" gravel roads (possibly with a couple water crossings!), but since it's not a race, riders of any speed should feel welcome. The easier route along a main highway has one large but gentle grade (and a few smaller hills) and is all paved. The drawback of the easy route is that traffic, particularly loud motorcycles, can become tiresome. The suggested route is fantastic and quiet, and should be doable for any halfway fit cyclist. Again, we're not talking whippet-thin racer-boy/girl fitness, but you may become familiar with the low gears on your bike. Single-speeders may be doing some pushing. We all have different levels of fitness and strength and speed, but who cares? This will not be a day to establish the pecking order, but to enjoy the scenery, our bicycles, and our company. The custom will be to regroup at the tops of hills and at intersections and to help each other and keep the group together as much as possible. Nobody should have to ride alone.
The Wisconsin side of Lake Pepin offers numerous cafes and other food sources. Generally speaking, there is a little town with a decent restaurant or cafe every 7-10 miles or so. These towns are all along the highway. The main route will visit these towns, thereby merging with the easier route several times during the day. In other words, there are plenty of bail-out options for those who get tired and want an easier alternative.
By the "easy" route, Wabasha is about 40 miles from where we'll begin in Red Wing. By the suggested main route, it will be closer to 50-55 miles. I am planning to arrange for free camping in Wabasha at the city park, where showers are available. If that fails, there are a couple campground options not far from town. Wabasha has some hotel options, too. Americinn is the biggest place, but I have no specific recommendations. Anyway, non-campers will be responsible for making their own hotel arrangements for Saturday, June 2, in Wabasha. Across the river in Nelson, WI, is the Nelson Cheese Factory, which is my favorite restaurant around there, but Wabasha has a couple dining options, too. Wabasha also has a large-ish grocery store for those who'd like to roll their own dinner and/or breakfast.
Sunday, June 3, we will ride the Minnesota side of the lake back to Red Wing. Again, there will be the suggested main route and an alternate easy route, with a couple merge points. The main route heads up the bluff for a long climb out of Wabasha. Later, the descent into Lake City is thrilling and gorgeous. Lake City is a good place for lunch, with a number of restaurants and stores. From Lake City, the main route starts on a smooth, easy gravel road, and later a long paved climb and subsequent descent into Red Wing. The easy route along the highway is flatter and all paved, but traffic can be tiresome. Total mileage is comparable to that of day 1, possibly somewhat shorter.
Back in Red Wing, I assume people will scatter. Maybe some will get together for a drink and a snack, and maybe some will hit the road for home immediately.
Tuesday, February 7, 2012
3 comments:
- James Fisher said...
Still on a post surgery restriction but if I am released to ride in the next month or so and able to get myself back to at least a partial condition after 6 months, count me in!
- February 8, 2012 at 6:55 PM
Could you clarify a couple of issues for me? What will the entry fee be? Also, I do not own a Rivendell (beyond my budget, but I subscribe to their ideals).
Are other bike brands acceptable?
Thanks for arranging the plans Jim.
- February 9, 2012 at 6:54 AM
- Jim Thill said...
No entry fee, but you'll need money for food, lodging/camping, and other expenses. All bikes are welcome.
- February 9, 2012 at 11:46 AM
Subscribe to: Post Comments (Atom)


text:
Israel News - Stay Informed, Pray Informed
America's embassy in Tel Aviv conspicuously raised a rainbow-colored flag in support of this week's "gay pride" festivities in the Israeli coastal city.
US Ambassador to Israel Dan Shapiro was not shy about the fact that "for the first time in history the US Embassy in Tel Aviv has raised the Pride flag together with our American flag. We are proud to join with the municipality of Tel Aviv-Yafo and its residents in celebrating LGBT Pride Week."
The week-long festivities are an annual occurrence in Tel Aviv, which has taken to publicly advertising itself as the Middle East's most gay-friendly city and a desirable travel destination for the homosexual community worldwide.
In 2010, the Tel Aviv Municipality and Israel's Ministry of Tourism invested a combined $88 million in a massive advertising campaign in England and Germany aimed at branding Tel Aviv as a leading international gay vacation spot.
In a 2011 survey conducted by the gay travel website Gaycities.com, respondents agreed by voting Tel Aviv as the top gay travel destination for that year.
PHOTO: US Ambassador to Israel Dan Shapiro (center) taking part in Tel Aviv's Gay Pride festivities
Want more news from Israel?
Click Here to sign up for our FREE daily email updates
For Information & Reservations:
E-Mail: firstname.lastname@example.com
Israel Today
Free to Your Inbox


text:
Locate the power button. This is the rectangular button on the top left side of the iPod Touch.
Locate the home button. This is the circular button on the face (screen) of your iPod towards the bottom.
Hold the home button and the power button at the same time and wait until the device shows the Apple logo on your screen (should take around 30 seconds). Your iPod should reboot with your files just the way you left them.
If the hard reboot doesn't solve your problem then connect your iPod to iTunes. iTunes should detect a problem with your iPod and give you an option to restore it to factory settings (erases all your songs and files so back them up first).


text:
Join more than 2,000 program directors in using ACTIVE's software and marketing to manage and grow your campTell Me More
ACTIVE's Camp Manager is an all-in-one online registration, camp management and marketing software solution. Whether you run overnight summer camps, day camps, sports camps and clinics, art classes, educational camps, or other types of youth programs, Camp Manager's cloud-based software is at your side to lighten your load and let you make smart, data-driven decisions while also helping you deliver great customer service.
Manage camps and programs with responsive, intuitive camp software. Create camp forms that are a breeze to complete and delight your camper families.
Work wherever and whenever you need to. Access your camp info on your smart phone or tablet. Let families register their children 24/7, even on the go.
Grow online camp registrations with email templates, integrated social media and your ACTIVEkids listing. Get the latest branding, website, SEO and marketing tactics from our expert consultants.
Modernize your registration process and attract customers with sleek online camp registration forms.
Transform the way you run your programs with a camp management hub you can access from anywhere, anytime.
Automate and stand out by using modern marketing and communication tools that are integrated with your camp software.
Camp forms are perfect for managing all camp types from summer camp registration to sports camps, church camps, and more. Let ACTIVE Camps help you efficiently manage your campers.


text:
DOLPHIN-spotting boats operating from the Cromarty Firth may pose the greatest risk to the Moray Firth's resident population of 200 bottlenose dolphins, a new report for Scottish Natural Heritage has warned.
Routine traffic – such as shipping associated with offshore renewables fabrication facilities in the area – is less likely to have an adverse effect on the dolphins than unregulated vessels that seek out the mammals.
Scientists believe that the small and isolated nature of the dolphin population makes them more prone to being disturbed by the tour boats that target foraging hotspots such as the Chanonry Narrows, the mouth of the Cromarty Firth and the Kessock Channel. It appears that the dolphins are less perturbed by vessels that simply pass through, according to researchers.
Experts at Aberdeen University developed a computer model to help predict the effects on dolphins from proposed developments in the Moray Firth area, as well as tour boats and other marine traffic.
The SNH report stated that a lack of information on the likely consequences of marine and coastal developments on the dolphin interest of the Moray Firth special area of conservation (SAC) had acted as a constraint in the past on the public bodies responsible for granting or advising on such developments, resulting in delays in the processing of planning permissions, marine licences or harbour revision orders, "which in turn could hold up important development opportunities for the area".
The report said that the computer modelling had yielded useful findings on the effect of shipping on dolphins. "It has shown that routine traffic is less likely to have an adverse effect on the dolphins than vessels which specifically target them – unregulated tour boats or recreational craft."
It concluded that foraging hotspots should be considered sensitive to disturbance by boats and that such activity should be kept to a minimum in such areas. However, there was less need for restrictions in areas where boating activity was predictable and where it did not seek contact with the animals.
Ben Leyshon, of SNH, said: "We aim to produce a tool that can inform future development while recognising we have a duty to protect the wild mammals in our waters."
George Farlow, vice-chairman of Highland Council's planning, environment and development committee, said: "This is an important piece of work which will support future sustainable development within the Moray Firth, while ensuring that we afford bottlenose dolphins the protection they deserve."


text:
Check out OpenSSL Notification
OpenSSL, and therefore, maybe some of your programs that depend on OpenSSL for security (Apache, OpenSSH, etc. etc.) may also be vulnerable if they are linked statically.
This is a very serious problem, and I suggest downloading the newest source asap.
Check this out and update your machines.
I haven't found a published exploit yet, but I don't think that is a good idea to wait for it.
Anyway, happy compiling.
Remember, if you aren't using dynamic linked files, recompile any programs that use the SSL libraries.
If you are running a web server, update as soon as possible. It seems an obvious target, since it is protected, etc.
If you have questions on recompiling, if you are an RPM installer, just ask.
Here's the quick and dirty install method
sh-2.05b# mkdir /usr/local/src
sh-2.05b# cd /usr/local/src
sh-2.05b# wget http://www.openssl.org/source/openssl-0.9.7c.tar.gz
sh-2.05b# tar -xzvf openssl-0.9.7c.tar.gz
sh-2.05b# ln -s openssl-0.9.7c openssl
sh-2.05b# cd openssl
sh-2.05b# ./config --prefix=/usr/local/openssl
sh-2.05b# make
sh-2.05b# make depend
sh-2.05b# make install
sh-2.05b# vi /etc/ld.so.conf
* add /usr/local/openssl/lib to this file
sh-2.05b# ldconfig
* Then recompile all the other applications that use this.
If you are using RPM or binary installations, get the source for those programs and run
sh-2.05b# ./configure --help | less
in the directory you untar them into.
Read each option, and be sure to be consistent.
Most applications will figure out what you have and use that, but be SURE to point to the correct installation of OpenSSL.
This is usually ./configure --openssl=/usr/local/openssl ..other switches
Have fun
You may need to recompile your other applications to use this.


text:
On Sunday, I preached my first sermon in my new role as Assistant Minister at St Augustine's Neutral Bay.
Recent blog posts
- So here's the plan (Ephesians 1:8–10)February 19, 2019The God of the universe has a plan to sum up all things in Christ. He's revealed that plan to us, and is putting it into effect through the gospel.
- Are you trying to redeem yourself? (Ephesians 1:6b–7)February 15, 2019When it comes to God, we can't redeem ourselves. Redemption is something that God has to do—and has done—for us. This is why it's such a wonderful thing.
- What if it's not about me? (Ephesians 1:4–6a)February 12, 2019The Bible gives us a vision for living life that's the exact opposite of the narcissistic attitude. This is highly confronting, and profoundly comforting.
- Overview of Romans 1–5: PowerPointFebruary 10, 2019An overview of the logic of Romans 1–5 (PowerPoint file)
- #inChrist (Ephesians 1:3)February 8, 2019Ephesians starts with an explosion of praise for the 'blessings' God has given to us. But they're different from the kind of blessings we often talk about.
- This God (Ephesians 1:2)February 5, 2019Many people believe in 'God'. But the word 'God' means different things to different people. What does the Bible say about God: who he is and what he's like?
- Amazing holiness (Ephesians 1:1b)February 1, 2019God sees believers in Jesus as holy. This fact is amazing, especially when you grasp the history of the gospel going out from Israel to the nations.
- Paul: in his own words (Ephesians 1:1a)January 29, 2019Who do you think the Apostle Paul is? It's worth listening carefully to the words Paul himself chooses to define his own identity at the start his letter.
- Lift Your Eyes: Introducing EphesiansJanuary 25, 2019When believers in Jesus were in danger of losing heart, Paul wrote Ephesians. This letter lifts our eyes to see God's amazing plan through Jesus Christ.
- From temple to meat market in ancient PompeiiJanuary 7, 2019A very quick journey from the temple of Jupiter in Pompeii to the meat market. This helps us to understand 1 Corinthians 8-10 and probably also Romans 14.
On this site
1 Corinthians 1 Timothy Acovenantalism Series Atonement Bible talks Biblical theology biblical word power Church Church History Covenant Creation Death Environment Ephesians Epistemology Eschatology Ethics Evangelism Faith Forgiveness Galatians General GoThereFor Greek Hebrew Isaiah Jewish Identity Justification Laughing on Inside Law LiftYourEyes Ministry Mission Moore College Paul Paul and the Vocation of Israel Psalms Revelation Romans Sexuality Speech and Salvation Sustainable Sacrifice Wisdom Women Sermons and the Bible wordworks-isaiah


text:
The NaturePlus Forums will be offline from mid August 2018. The content has been saved and it will always be possible to see and refer to archived posts, but not to post new items. This decision has been made in light of technical problems with the forum, which cannot be fixed or upgraded.
We'd like to take this opportunity to thank everyone who has contributed to the very great success of the forums and to the community spirit there. We plan to create new community features and services in the future so please watch this space for developments in this area. In the meantime if you have any questions then please email:
Fossil enquiries: email@example.com
Life Sciences & Mineralogy enquiries: email@example.com
Commercial enquiries: email@example.com
We moved into a 1930's house a few months back in the countryside and all was well. However, about 9 days ago, these little bugs appeared in the bathroom on the bathroom window sill.
At first I thought it was just dust, as they are really tiny (about 1mm I'd guess). However I saw them moving slightly so investigated further. They're black (although some (albeit a few only) are very, very white, no other colours I can see, might be two different things, the white ones are impossible to make out), very small, hardly move at all, don't fly and don't jump.
At first I thought they were baby spiders, but not after taking the photograph (attached).
I then found them all over the bath, up the walls, over the ceiling a little and over the tiled shelf and, eventually, the floor. They mostly seem to like the white goods in the bathroom, but also the corners of the room.
As of Saturday-Friday last week, every time I wet a piece of tissue and run it around the bathroom floor along the edges, I would collect about 1000 of them. I'm estimating that based on wetting one sheet of paper, doing a small area and then counting, as best I could, how many were on the sheet of paper. By the time I got only 1/4 of the way round, the paper was almost black. I did this twice a day.
I sprayed a Raid beetle and spider spray but they didn't seem to die (it did seem to kill some, but then some were still happily moving).
However since Saturday I've been clearing the bathroom of about 200-300 a day, rather than a couple of thousand. So their numbers appear to be declining.
We have recently had new windows installed, so they may have been disturbed during that, but that was three weeks ago and they only showed up just over one week ago.
They've also appeared in the next door bedroom, again on the white plastic window sill. I haven't found them anywhere else, but because they're so small, I doubt I would really.
Any help greatly appreciated, as they're getting all over the flannel, hand towels, shaving equipment, bath (at it's worst, they were crawling over both top edges of the bath, to the point that it looked pebbledashed with black paint), etc. etc.
The photo isn't great (especially the one I have zoomed in on), but I can't stress enough how small they are, and I couldn't get a better picture than this.
From the description of your property, can I hazard a guess that you have (or had) swallows or other birds nesting under the eaves or in the attic? If not birds, any rodent, bee or wasps nests?
I would definitely wait for one of our experts to give their opinion but to my untrained eye it looks like it could be a mite. The work performed on your windows could have disturbed a nest in the attic leading to the mites migrating into the house.
E.g. see all the responses to this thread:
And this thread:
Btw the following link that provides information about bird mites is in the second thread in my reply:
Thanks Jonathan
I will wait as you suggest to see if somebody can confirm, but what you've suggested does sound right.
Up to about two weeks ago, we had Starlings (spelling?) nesting in the eaves of the bathroom. We could hear them, but we stopped being able to hear them about two weeks ago, so suspect they flew then. It would have been a few days after that that these bugs appeared.
So...that all makes sense. I can't work out where they're coming in from, as I can't even find a crack, so I'm wondering whether they're coming in under the bath behind the bath panel that is sealed on with sealant (not our doing, previous owners). I'll cut around that this weekend to see if that's where the little bug...ers...are coming in.
Any suggestions with what to use to get rid of them? I don't really want to call in pest control and go to all that expense unless I have to.
Really appreciate your response!
I agree with Jonathan - Tropical fowl mite, Ornithonyssus bursa, is a common mite carried by starlings and often infest the nest after the adults and chicks have left/fledged. Fortunately they cannot sustain themselves by feeding on our blood full time but can and will try feeding on us and are more than a nuisance why this occurs. I feel that a dose of mite powder (as sold for chickens) could solve this problem.
Thanks, that helps.
Being a complete newbie to this (used to be a town person, now a newcomer to the countryside) where would I likely find something like Mite Powder? I'm assuming a pets at home or Homebase/supermarket type place is out of the equation?
And would I just dust that all around where I can see them residing. or should I look to discover where they're coming in (I don't really fancy getting up to the eaves...they are VERY high (it's a very tall, old, house).
Should I go to the trouble of cutting the sealant around the bath panel and investigating under there, or just dust it around the bath, window sill and edges of the floor all around the bathroom and leave it at that?
We don't use that room other than to clean teeth at the end of the night, as the shower room downstairs gets used for washing, etc. So not worried about leaving the powder down for days/weeks at a time if need be to get rid of them.
this link will tell you a bit about mite migration - assuming you do have a mite flavoured invasion!! It seems moisture is the key - at least for the house mite because they can't drink - but whatever type of mite you might have, this problem might not clear up so quickly unless you can tackle the steamy moist goodness of a typical bathroom environment. I would thoroughly suggest a strong dehumidifier to counteract the effect if showers, hand and face washing etc. (probably why they like your towels so much!)
But be prepared for them to try and find somewhere else if you bathroom gets drier..
Good luck!
look at www.tinywhitemites.co.uk


text:
NEW YORK (MarketWatch) — Hostess Brands Inc. has seen a "flood of inquiries" in its brands, the company said during a bankruptcy-court hearing on the snack maker's plan to liquidate and cut thousands of jobs, following the failure of last-ditch mediation.
In a bitter end for the maker of Twinkies and Wonder Bread, the U.S. Bankruptcy Court for the Southern District of New York approved the company's motion "for the orderly wind-down of its business and sale of its assets."
Hostess Brands said it intends to retain approximately 3,200 of its employees to assist with scaling down operations, in a process expected to take a year.
Hostess will close 33 bakeries, 565 distribution centers, about 5,500 delivery routes and 570 bakery outlet stores, as well as cut 18,500 jobs.
"Employee head count is expected to decrease by 94% within the first 16 weeks of the wind down," according to the company.
Ahead of the hearing in bankruptcy court in White Plains, N.Y., Hostess Chief Executive Gregory Rayburn told Bloomberg TV that the Irving, Texas-based company has received interest in buying some of its brands from rival food makers and private-equity firms.
But he warned that any buyer will be unlikely to pick up the entire company and keep all its operations open, in a blow to job prospects for Hostess workers.
Meanwhile, shares of Flower Foods Inc. FLO, -0.05% rose 3.4% Wednesday on speculation that the maker of Nature's Own bread and snack cakes may bid for Hostess assets. In April 2011, Flower Foods bought Tasty Baking Co. for $165 million in cash.
Precipitating the liquidation plans, Hostess failed to reach a contract agreement with the Bakery, Confectionery, Tobacco and Grain Millers Union, which represents 5,600 workers at the company.
In a brief statement late Tuesday, the company said mediation proved unsuccessful. Bankruptcy Court Judge Robert Drain had asked the two sides to meet Tuesday prevent a shutdown of the 82-year-old business.
Hostess chief Rayburn said some members of the bakers' union had heard that Mexico's Grupo Bimbo SAB de CV BIMBOA, +0.08% would buy the company. However, such a transaction could not gain antitrust approval because that firm recently closed on the purchase of bakery assets from Sara Lee Corp., he added.
The Teamsters union had approved a pay package with Hostess, but the bakers' union didn't go along with it.
"Demand hasn't been our problem. ... The problem was always the cost structure," Rayburn said. "We gave [it] every shot we could. The lenders tried to do everything they could. The Teamsters did everything they could to give this company a chance to survive. We just couldn't convince the bakers."
In a nod to the intense media coverage and hype around the demise of the treat maker, Rayburn said he heard of boxes of Twinkies were selling for $5,000 on eBay. Read more on Hostess reaction: How to stockpile Twinkies.
New York Times columnist Paul Krugman published his "Twinkie economic manifesto" in a tribute to the "iconic snack."
The sugary confection broke into pop culture in the 1950s, when Hostess sponsored TV's "Howdy Doody Show."
The fondness for Hostess extended beyond baby boomers. Comedian Jimmy Fallon joked that Pabst Blue Ribbon beer may buy Hostess and start making Twinkies. "Yeah, beer and Twinkies — or as I called that in college, 'brunch,'" Fallon wrote on Twitter.


text:
The catenin family of peripheral cytosolic proteins (consisting of three major species) has been determined to bind selectively to the highly conserved cytoplasmic tail domain of the cell-to-cell adhesion cadherin proteins. One of these constituents, beta-catenin, binds directly to the E-cadherin cytoplasmic domain and plays a critical role in intercellular adhesion and signal transduction between neighboring cells. Because beta-catenin has also been determined to form complexes with the tumor suppressor gene product, APC, the protein is also suspected to be important in tumorigenesis. In addition, beta-catenin may be involved in the regulation of gene activity due to its constitutive interaction with transcription factors from the TCF/LEF gene family.
The technique of double immunofluorescence was employed to simultaneously label an adherent culture of rat thoracic aorta (A-10 line) cells (illustrated above) with mouse anti-histone and rabbit anti-beta-catenin primary antibodies, followed by goat anti-mouse and anti-rabbit secondary antibodies conjugated to Marina Blue (histones) and Rhodamine Red-X (beta-catenin), respectively. The culture was counterstained with Alexa Fluor 488 conjugated to phalloidin, targeting the intracellular filamentous actin network. Images were recorded in grayscale with a QImaging Retiga Fast-EXi camera system coupled to an Olympus BX-51 microscope equipped with bandpass emission fluorescence filter optical blocks provided by Omega Optical. During the processing stage, individual image channels were pseudocolored with RGB values corresponding to each of the fluorophore emission spectral profiles.
© 1995-2019 by Michael W. Davidson and The Florida State University. All Rights Reserved. No images, graphics, software, scripts, or applets may be reproduced or used in any manner without permission from the copyright holders. Use of this website means you agree to all of the Legal Terms and Conditions set forth by the owners.
This website is maintained by our