Indole inhibitors of human nonpancreatic secretory phospholipase A2. 1. Indole-3-acetamides.

Phospholipases (PLAs) produce rate-limiting precursors in the biosynthesis of various types of biologically active lipids involved in inflammatory processes. Increased levels of human nonpancreatic secretory phospholipase A2 (hnps-PLA2) have been detected in several pathological conditions. An inhibitor of this enzyme could have therapeutic utility. A broad screening program was carried out to identify chemical structures which could inhibit hnps-PLA2. One of the lead compounds generated by the screening program was 5-methoxy-2-methyl-1-(phenylmethyl)-1H-indole-3-acetic acid (13a). We describe the syntheses, structure--activity relationships, and pharmacological activities of a series of indole-3-acetamides and related compounds derived from this lead. This SAR was undertaken with the aid of X-ray crystal structures of complexes between the inhibitors and hnps-PLA2 which were of great value in directing the SAR.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 2. Indole-3-acetamides with additional functionality.

As reported in our previous paper, a series of indole-3-acetamides which possessed potency and selectivity as inhibitors of human nonpancreatic secretory phospholipase A2(hnps-PLA2) was developed. The design of these compounds was based on information derived from x-ray crystal structures determined for complexes between the enzyme and its inhibitors. We describe here the further implementation of this structure-based design strategy and continued SAR development to produce indole-3-acetamides with additional functionalities which provide increased interaction with important residues within the enzyme active site. These efforts led to inhibitors with substantially enhanced potency and selectivity.

Indole inhibitors of human nonpancreatic secretory phospholipase A2. 3. Indole-3-glyoxamides.

The preceding papers of this series detail the development of functionalized indole-3-acetamides as inhibitors of hnps-PLA2. We describe here the extension of the structure-activity relationship to include a series of indole-3-glyoxamide derivatives. Functionalized indole-3-glyoxamides with an acidic substituent appended to the 4- or 5-position of the indole ring were prepared and tested as inhibitors of hnps-PLA2. It was found that the indole-3-glyoxamides with a 4-oxyacetic acid substituent had optimal inhibitory activity. These inhibitors exhibited an improvement in potency over the best of the indole-3-acetamides, and LY315920 (6m) was selected for evaluation clinically as an hnps-PLA2 inhibitor.

Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum.

The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum. Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis. The culture response was assessed chemically and morphologically, using computerised image analysis. High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis. However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss. Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process. Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P. chrysogenum. By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P. chrysogenum.

Incorporation of 14C-labeled compounds into sinefungin (A9145), a nucleoside antifungal antibiotic.

Streptomyces griseolus produces a complex of antifungal nucleoside antibiotics that contain an ornithine residue linked to the ribose moiety of adenosine. 14C-Labeled compounds were added to cultures of S. griseolus (approximately 0.5 muCi/ml culture broth) and the amount of label incorporated into the two major antifungal components (sinefungin and factor C) was measured. Substantial incorporation (16 approximately 52%) was obtained with adenosine [8-14C], ATP [14C(u)], adenine [8-14C], L-ornithine [14C(u)], and DL-citrulline [5-14C]. Glycine glucose L-arginine, and acetate were incorporated to the extent of 1.7 approximately 4.7%. Studies were conducted on the fermentation time course and on the time dependence of label incorporation in order to optimize the incorporation of labeled adenine into sinefungin. Adenine [8-14C] incorporation and sinefungin specific activity were highest 48 hours after label addition and both declined during subsequent incubation. As much as 43% of the labeled adenine was incorporated into the antibiotic and sinefungin was produced with a specific activity of 24.8 muCi/mg. The labeling experiments suggest that a performed adenine derivative (e.g., an adenine nucleotide) and ornithine (or a closely related metabolite) are direct biosynthetic precursors of sinefungin.

Preparation and evaluation of 3,4''-ester derivatives of 16-membered macrolide antibiotics related to tylosin.

A large group of ester derivatives of tylosin-related macrolides was prepared in which the hydroxyl groups at C-3 and C-4'' were acylated by either chemical or biochemical methods. Most of the derivatives exhibited excellent in vitro antimicrobial activity. However, only the 3,4''-diacyl derivatives of tylosin and macrocin showed any significant improvements of in vivo efficacy against experimental infections in rodents.

Predictive spelling with a P300-based brain-computer interface: Increasing the rate of communication.

This study compared a conventional P300 speller brain-computer interface (BCI) to one used in conjunction with a predictive spelling program. Performance differences in accuracy, bit rate, selections per minute, and output characters per minute (OCM) were examined. An 8×9 matrix of letters, numbers, and other keyboard commands was used. Participants (n = 24) were required to correctly complete the same 58 character sentence (i.e., correcting for errors) using the predictive speller (PS) and the non-predictive speller (NS), counterbalanced. The PS produced significantly higher OCMs than the NS. Time to complete the task in the PS condition was 12min 43sec as compared to 20min 20sec in the NS condition. Despite the marked improvement in overall output, accuracy was significantly higher in the NS paradigm. P300 amplitudes were significantly larger in the NS than in the PS paradigm; which is attributed to increased workload and task demands. These results demonstrate the potential efficacy of predictive spelling in the context of BCI.

Inhibition of Candida albicans by Lactobacillus acidophilus: evidence for the involvement of a peroxidase system.

A range of cultures of Lactobacillus acidophilus was isolated from patients using oral, vaginal and endocervical swabs. These were investigated for their ability to (1) inhibit the growth of Candida albicans, and (2) generate peroxidase, hydrogen peroxide and hypothiocyanite. Inhibition of Candida albicans and hydrogen peroxide production was detected in nine out of twelve strains whereas peroxidase production was only detected in three out of twelve strains, all from oral swabs. Hypothiocyanite production was detected in two strains and it was only detected in these strains after growth in MRS medium in aerobic conditions.

Isolation and characterization of a unique endo-beta-1,6-glucanase from the yeast Saccharomycopsis fibuligera NCYC 451.

A beta-glucanase enzyme has been described which has beta-1,6 activity but no beta-1,3 activity. It was isolated and purified from cell free extract and culture free medium of Saccharomycopsis fibuligera by a combination of techniques that included adsorption on DEAE-Sepharose and gel filtration through a Sephacryl S-300 column. The extracellular endo-beta-1,6-glucanase had similar physicochemical properties to those of the intracellular one. The intracellular enzyme behaved as an acidic protein with pI 3.95. It had an optimum pH of 5.5 and optimum temperature of 50 degrees C. The enzyme was specific for beta-1,6-glucosidic linkages by an endo-acting mechanism. The molecular weight of the intracellular enzyme was estimated at 51 kD from gel filtration compared with 43 kD for the extracellular enzyme.

The inhibition by high concentrations of (2-chloroethyl)-trimethylammonium chloride (CCC) of chlorophyll and protein synthesis in excised barley leaf sections.

CCC at concentrations of 10(-3) M and higher inhibits chlorophyll synthesis and (3)H-leucine incorporation into a protein fraction by barley leaf sections. Neither of these effects is reversed by exogenous GA3. No effect of CCC was observed on oxygen uptake by the leaf sections. The results indicate that high concentrations of CCC may act through an inhibition of protein synthesis, rather than through a direct effect on endogenous gibberellin production.

Isolation and partial characterization of yeast mannan hydrolysing enzymes from bacterial isolates.

A range of aerobic, mesophilic bacterial strains capable of producing extracellular alpha-mannanase and alpha-mannosidase enzymes were isolated from various natural habitats using yeast cell wall material as the selective medium. Five of them were capable of producing extracellular alpha-mannosidase and alpha-mannanase on yeast mannan as the sole carbon substrate, and they were tentatively identified to the genus level as Arthrobacter, Streptomyces, Pseudomonas, Bacillus and Acinetobacter. Among these strains, Arthrobacter M-02 showed the highest biosynthetic activity of the enzymes. The crude enzyme preparations from the strains were active at pH 4 to 9, reaching a maximum activity between 5.5 and 6.8. The crude Arthrobacter M-02 alpha-mannosidase was more thermostable than that of the other strains, but the alpha-mannanase of the strain was thermolabile.

The use of step enzymes as markers during meiosis and ascospore formation in Saccharomyces cerevisiae.

The activities of ornithine aminotransferase, sucrase and acid and alkaline phosphatases have been studied throughout sporulation in Saccharomyces cerevisiae. The same enzymes were monitored during synchronous vegetative growth. Each of these enzymes has been demonstrated to increase in a 'step' manner during both growth and sporulation. Alkaline phosphatase increased in a two-step manner whereas the others increased in a single step. The times of increase of these enzymes formed a similar sequence during both sporulation and growth. It has been proposed that these enzymes are under a common mechanism of control during growth and sporulation and that the sequence of enzyme appearance may be used as markers of the sporulation process.

Evidence for the involvement of thiocyanate in the inhibition of Candida albicans by Lactobacillus acidophilus.

Lactobacillus acidophilus has been found to inhibit Candida albicans when grown on MRS agar plates. Attempts to isolate an active factor responsible for this inhibition from liquid culture and agar plates were not successful. The addition of sodium thiocyanate to the agar was found to increase the inhibition offered by the lactobacillus. The results indicate that hydrogen peroxide produced by the lactobacillus is being used to convert the thiocyanate to hypothiocyanate which is more toxic. The involvement of a lactobacillus peroxidase in this conversion is postulated.

Discrimination by heat and proteinase treatments between flocculent phenotypes conferred on Saccharomyces cerevisiae by the genes FLO1 and FLO5.

The effects of elevated temperature and of digestion with a variety of proteinases on the flocforming ability of flocculent strains of Saccharomyces cerevisiae, both genetically defined (FLO1 and FLO5) laboratory and genetically undefined brewing strains, have been determined. This has permitted classification of the flocculent phenotypes of these strains according to criteria other than quantitative grading of flocculence. The flocculent phenotypes conferred by both the FLO1 and the FLO5 gene were irreversibly lost upon treatment with pronase, proteinase K, trypsin or 2-mercaptoethanol treatments. However, the floc-forming ability of cells of the FLO1 strain ABXL-1D was destroyed by chymotrypsin digestion and was stable to incubation at 70 degrees C, whereas the floc-forming ability of cells of the FLO5 strain ABXR-11A was resistant to the action of chymotrypsin and was heat labile. Tetrad analysis of a cross of these FLO1 and FLO5 strains indicated that the chymotrypsin and heat sensitivity phenotypes were FLO-gene determined. It appears that expression of the FLO1 and FLO5 genes leads to the production of different and characteristic cell-wall proteins underlying their respective flocculent phenotypes.

Role of proteases in autolysis of Penicillium chrysogenum chemostat cultures in response to nutrient depletion.

An industrial strain of Penicillium chrysogenum was subjected to carbon or nitrogen limitation in a chemostat and the response monitored in terms of the "classical" indicators of autolysis (biomass decline and ammonia release), culture degradation (as measured by image analysis) and by obtaining profiles for three classes of proteases implicated in autolysis. Under both sets of conditions (carbon or nitrogen limitation), once started, autolysis involved a succession of different protease activities. The first stages of the process of autolysis in starved chemostat cultures was associated with peaks in the activities of both serine and aspartyl proteases, coinciding with the mobilisation of endogenous energy reserves. Conversely, a peak in the activity of metalloproteases was associated with the later stages of autolysis, perhaps occurring in response to depletion of endogenous energy reserves; the activity of these enzymes led to gross culture degradation, disintegration of ordered mycelial structures and signalled the end of metabolic activity (respiration) within the culture. These findings indicate that strategies intended to control/regulate autolysis in large-scale industrial fungal cultures might profitably be focused on regulation of the activity of key classes of proteases involved in the series of events leading to culture degradation.

Two staining methods for selectively detecting isomaltase and maltase activity in electrophoresis gels.

Two methods for specifically detecting maltase, alpha-glucosidase, or isomaltase activity in electrophoresis gels are described. Both systems couple the formation of glucose by enzyme action on maltose or isomaltose to the generation of a colored product. System A uses an agarose overlay which contains substrate, glucose oxidase, peroxidase, 2,4-dichlorophenol, and 4-L-amino-phenazone. A purple color is produced at the site of enzyme activity. No hazardous chemicals are used at any stage. The stain is simple, rapid, sensitive, and inexpensive and does not interfere with subsequent protein staining. However, the stain is not permanent. System B was developed to give a permanent stain. The gel is overlaid with agarose containing substrate, glucose oxidase, phenazine methosulfate, and nitroblue tetrazolium. Glucose production results in the nitroblue tetrazolium being oxidized to an insoluble formazan with a dark blue color. This stain is also sensitive, rapid, and inexpensive but does use hazardous chemicals and if overstaining occurs this can interfere with subsequent protein staining. Neither system inactivates the localized enzymes which can be recovered from the gel if desired.

Measurement of autolysis in submerged batch cultures of Penicillium chrysogenum.

The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and beta-1, 3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in beta-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum.

Quantification of autolysis in Penicillium chrysogenum by semiautomated image analysis.

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.