Chemical validation of GPI biosynthesis as a drug target against African sleeping sickness

It has been suggested that compounds affecting glycosylphosphatidylinositol (GPI) biosynthesis in bloodstream form Trypanosoma brucei should be trypanocidal. We describe cell-permeable analogues of a GPI intermediate that are toxic to this parasite but not to human cells. These analogues are metabolized by the T. brucei GPI pathway, but not by the human pathway. Closely related nonmetabolizable analogues have no trypanocidal activity. This represents the first direct chemical validation of the GPI biosynthetic pathway as a drug target against African human sleeping sickness. The results should stimulate further inhibitor design and synthesis and encourage the search for inhibitors in natural product and synthetic compound libraries.     

Diversity of the Clostridium coccoides group in human fecal microbiota as determined by 16S rRNA gene library

Fecal microbiota were analyzed in seven healthy individuals by 16S rRNA gene libraries (universal library) using universal primers, and the Clostridium coccoides group libraries using the universal primer 27F and the C. coccoides group-specific primer Erec482. The universal libraries were used in our previous studies. The 972 clones obtained from two different primer set libraries belonged to the C. coccoides group and were classified into 139 operational taxonomic units (OTU) (at least 98% sequence similarity). Of these, 41 OTU were detected commonly from universal libraries and C. coccoides group libraries. One hundred and ten OTU were detected from the C. coccoides group libraries. Fifteen new OTU were isolated from the C. coccoides group libraries in human gut. Most of the OTU did not correspond to known species, thus representing as-yet-uncultured bacteria. We also detected OTU that related to the butyrate-producing bacteria. The C. coccoides group consisted of an average of 35 OTU, although there were differences in the number and the type of species in each individual. When fecal microbiota were analyzed using universal libraries, the OTU belonging to the C. coccoides group detected in elderly individuals were fewer than those detected in adult individuals. When C. coccoides group libraries were used, the numbers of OTU in elderly and adult individuals were not different. Interindividual differences in the composition of OTU belonging to the C. coccoides group were also observed in fecal microbiota.     

Thyrotropin-releasing hormone (TRH) analogues show enhanced CNS selectivity because of increased biological stability

TRH exerts both endocrinological and neuropharmacological actions. Two analogues of TRH, Pyr-His-Mep . NH2 (L-trans-3-methylprolineamide) and Pyr-His-Dmp . NH2 (L-3,3-dimethylprolineamide) have been examined for their neuropharmacological and endocrinological effects. Comparisons of their ability to provoke hyperthermia in rabbits demonstrated that both analogues were more potent than TRH, but like the parent peptide had only a limited ability to cross the blood brain barrier. This conclusion was confirmed by whole body autoradiographical studies. In contrast both analogues had a similar potency to TRH with respect to the ability to provoke TSH release. It is concluded that the increased potency in neuropharmacological tests results from enhanced bioavailability to CNS sites and that a similar rationale can be used to explain the CNS selectively claimed in the literature for other analogues of TRH.     

Archaeal diversity in a terrestrial acidic spring field revealed by a novel PCR primer targeting archaeal 16S rRNA genes

The phylogenetic diversity of archaeal 16S rRNA genes in a thermoacidic spring field of Ohwakudani, Hakone, Japan, was investigated by PCR-based analysis using a novel Archaea-specific primer designed in the present study. Clone libraries of archaeal 16S rRNA genes were constructed from hot water (78 °C) and mud (28 °C) samples by PCR using a newly designed forward primer and a previously reported forward primer with reverse primers. Most phylotypes found in the libraries from the hot water sample were related to cultured (hyper)thermophiles. The phylotypes and their detection frequencies from the hot water sample were similar for the libraries amplified with the two different primer sets. In contrast, phylotypes having a low similarity (<95%) to cultured Archaea were found in the libraries from the mud sample. Some of the phylotypes were relatively close to members of Thermoplasmata (80-93% similarity) and the others were not clearly affiliated with Crenarchaeota and Euryarchaeota, but related to Thaumarchaeota and Korarchaeota. The phylotypes and their detection frequencies were significantly different between the two libraries of the mud sample. Our results from the PCR-based analysis using the redesigned primer suggest that more diverse, uncultured Archaea are present in acidic environments at a low temperature than previously recognized.     

Synthesis of nucleoside libraries on solid support. II. 2,6,8-Trisubstituted purine nucleosides using 8-bromoguanosine as precursor

A series of 2,6,8-trisubstituted purine nucleoside libraries was prepared by parallel solid-phase synthesis using 8-bromoguanosine as a common synthetic precursor. Polystyrene-methoxytrityl chloride resin was linked to the N2 or O5' position of the guanosine analogues. 8-Bromoguanosine was derivatized at the C8 position via carbon-carbon bond formation. Nucleophilic aromatic substitution at C2 and/or C6 positions with various amines produced two series of purine nucleoside libraries with very diverse substitution.     

Enantioselective actions of 4-amino-3-hydroxybutanoic acid and (3-amino-2-hydroxypropyl)methylphosphinic acid at recombinant GABA(C) receptors

The R- and S-enantiomers of 4-amino-3-hydroxybutanoic acid (GABOB) were full agonists at human recombinant rho1 GABA(C) receptors. Their enantioselectivity (R>S) matched that reported for their agonist actions at GABA(B) receptors, but was the opposite to that reported at GABA(A) receptors (S>R). The corresponding methylphosphinic acid analogues proved to be rho1 GABA(C) receptor antagonists with R(+)-CGP44533 being more potent than S(-)-CGP44532, thus showing the opposite enantioselectivity to the agonists R(-)- and S(+)-GABOB. These studies highlight the different stereochemical requirements for the hydroxy group in these analogues at GABA(A), GABA(B) and GABA(C) receptors.     

Substrate spectrum of tyrocidine thioesterase probed with randomized peptide N-acetylcysteamine thioesters

Apparent kinetic constants k(cat) and K(m) were determined for tyrocidine thioesterase (TycC TE) using randomized peptide N-acetylcysteamine thioesters as substrate analogues. The enzyme has been found to be adequately active for the synthesis of positional-scanning libraries for novel antibiotic screening with reduced k(cat)/K(m) in the range of 2 to 82 folds lower than that of the wild-type sequence     

Methyllycaconitine analogues have mixed antagonist effects at nicotinic acetylcholine receptors

Bicyclic analogues of methyllycaconitine (MLA), such as 12, have been synthesised that incorporate the C1-OMe substituent present in the natural product. Electrophysiology experiments using Xenopus oocytes expressing nicotinic acetylcholine receptors (nAChRs) were conducted on these analogues and a related tricyclic analogue 2. The most potent compound, 2, was an antagonist at all receptors studied but displayed different antagonist effects at each receptor subtype. This study more clearly defines the biological effects of MLA analogues at nAChRs and demonstrates that these analogues are not selective ligands for the alpha7 nAChR subtype.     

Non-natural cinnamic acid derivatives as substrates of cinnamate 4-hydroxylase

Cinnamate 4-hydroxylase (C4H), a monooxygenase in the plant phenylpropanoid pathway, was assayed for its ability to hydroxylate 29 substrate analogues. Nine of the tested analogues with various aromatic side chains, including 3-coumaric acid, were metabolized by C4H. Seven products from these reactive analogues were characterized using LC/MS, 1H NMR and 13C NMR spectroscopic analysis. For example, caffeic acid was the product of 3-coumaric acid. The products 4-hydroxy-2-chlorocinnamic acid and 4-hydroxy-2-ethoxycinnamic acid are novel compounds that have not been previously reported. The kinetic parameters of C4H towards these analogues were determined.     

Synthesis and activity of phosphinic tripeptide inhibitors of cathepsin C

Phosphinic tripeptide analogues Gly-Xaapsi[P(O)(OH)CH(2)]-Gly have been developed as inhibitors of cathepsin C (DPP I), a lysosomal, papain-like cysteine protease. The target compounds were synthesised by addition of methyl acrylate to the appropriate phosphinic acids followed by the N-terminus elongation using mixed anhydride procedure. The latter step has been demonstrated to be a suitable method for N-terminal extension of the phosphinic pseudopeptide analogues without requirement of hydroxyphosphinyl protection. The title compounds appeared to be moderate inhibitors of the cathepsin C. However, although designed as transition state analogues, they surprisingly exhibited noncompetitive mode of binding to cathepsin C. Differences in kinetics of C-terminal acids and esters have been additionally observed.     

Steroid antagonism of the hypertensinogenic activity of adrenocortical steroids

Previous studies in sheep have provided evidence for a separate "hypertensinogenic" class of adrenocortical steroid activity which is not simply related to their classical mineralocorticoid (MC) and/or glucocorticoid (GC) actions. This study investigated the structure-activity relationships of the effects of structural analogues of prednisolone on mean arterial pressure (MAP), and MC and GC actions in sheep. Infusions of these synthetic GC at 0.6 and 24 mg/day produced variable pressor effects which were dissociated from their MC and GC actions. In other experiments, the minimum adrenocortical steroid requirement to reproduce the onset of ACTH-dependent hypertension was determined. Infusion of cortisol, aldosterone, 17 alpha-hydroxy progesterone and 17 alpha,20 alpha-dihydroxy-4-pregnene-3-one was found to be sufficient to reproduce the hypertensive response to ACTH administration in sheep. A subsequent experiment showed that substitution of cortisol by the more potent synthetic GC, prednisolone had no effect on MAP. Therefore, cortisol appears to exert an essential action in ACTH hypertension which is not dependent on its GC activity. Other studies have found that prednisolone (100 mg/day) antagonized 9 alpha-fluoro-prednisolone (0.6 mg/day) induced hypertension but not its MC effects. The effect of progesterone (500 mg/day) and the progesterone analogues, norethisterone, medroxy-progesterone and 16 alpha-methyl progesterone on ACTH (5 micrograms/kg per day) hypertension was investigated. Progesterone completely blocked the hypertension and MC effects of ACTH infusion, while medroxy-progesterone partially blocked the increase in MAP. These data support our concept of a "hypertensinogenic" class of steroid activity.     

Actions between neonicotinoids and key residues of insect nAChR based on an ab initio quantum chemistry study: hydrogen bonding and cooperative pi-pi interaction

Neonicotinoid insecticides show selective actions on insect nicotinic acetylcholine receptor (nAChR). Two key residues (Trp and Arg/Lys) have been identified as contributing to the neonicotinois binding. To investigate the selective mechanism, a computational model was set up to simulate the interaction between residues (Trp and Arg) of insect nAChR and neonicotinoids by quantum chemistry method. Three analogues of neonicotinoid derivatives without the chloropyridinyl moiety and 3-methyl-indole (3MI), guanidinium (Gua) were used to mimic the neonicotinoids and the side chain of key residues Trp and Arg accordingly. Interaction features of 3MI-analogues, analogues-Gua and 3MI-analogues -Gua complexes were analyzed comparatively. Hydrogen bonding between the nitro group of analogues and Gua was found to be the most important for binding. Moreover, the cooperative pi-pi interaction between analogues and the indole ring, which is strengthened by the existence of Gua, also contributes to the binding. The alternative binding model of neonicotinoids proposed here, although slightly different from others, might be close to the actual.     

Progress in chronophysiology of peptide hormones with emphasis on clinical application of analogues

It is now established that peptide hormones are among the most versatile substances involved in intercellular communications. By substitution of one or more natural aminoacids, numerous peptide analogues have been synthesized with increased duration of action and potency with respect to endogenous hormones. Most of these effects are programmable as a function of dose and timing of administration. Not surprisingly, the chronobiological use of hormone-related peptides may represent a valuable tool providing new approaches to diagnosis and therapy of different disease states. One pertinent example of the applicability of peptide analogues refers to a new short chain ACTH analogue (ACTH 1-17; Synchrodyn) that has been proved useful to reset or preset the rhythmic ordering of a number of functions and to provide, furthermore, information about specific pathogenetic mechanisms and/or subtle alterations of the adrenal function. A large body of evidence indicates that the same concepts apply to most actions of the gonadotropin-releasing hormone (LH-RH) related analogues. The efficacy of the so-called low-dose pulsatile LH-RH therapy in the management of hypogonadotropic hypogonadism and of some forms of infertility seems now established. The use of potent analogues is providing new approaches to the heretofore unsuccessful therapies in a number of disease conditions. In this light, chronoendocrinology and biotechnology have to be mutually supporting. Novel clinical applications are expected from the emerging use of portable instrumentation designed to record endogenous data and/or to deliver drugs according to a temporal program.     

Combinatorial synthesis of natural products

Combinatorial syntheses allow production of compound libraries in an expeditious and organized manner immediately applicable for high-throughput screening. Natural products possess a pedigree to justify quality and appreciation in drug discovery and development. Currently, we are seeing a rapid increase in application of natural products in combinatorial chemistry and vice versa. The therapeutic areas of infectious disease and oncology still dominate but many new areas are emerging. Several complex natural products have now been synthesised by solid-phase methods and have created the foundation for preparation of combinatorial libraries. In other examples, natural products or intermediates have served as building blocks or scaffolds in the synthesis of complex natural products, bioactive analogues or designed hybrid molecules. Finally, structural motifs from the biologically active parent molecule have been identified and have served for design of natural product mimicry, which facilitates the creation of combinatorial libraries.     

Beta-turns induced in bradykinin by (S)-alpha-methylproline.

The ability of (S)-alpha-methylproline (alpha-MePro) to stabilise reverse-turn conformations in the peptide hormone bradykinin (BK = Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) has been investigated. Two BK analogues containing alpha-MePro at position 3 or position 7 were synthesised and their conformations in aqueous solution investigated by NMR spectroscopy. Whereas BK is largely disordered on the NMR time scale both analogues showed ROE connectivities in 2D-ROESY spectra indicative of reverse-turn conformations at both Pro2-Phe5 and Ser6-Arg9, whose formation appears to be cooperative. Some potential applications of alpha-MePro as a reverse-turn mimetic in the construction of synthetic peptide libraries is discussed.     

Chymotrypsin substrate analogues inhibit endocytosis of insulin and insulin receptors in adipocytes

To explore the possible role of proteolytic step(s) in receptor-mediated endocytosis of insulin, the effects of inhibitors of various classes of proteases on the internalization process were studied in isolated rat adipocytes. Intracellular accumulation of receptor-bound 125I-insulin at 37 degrees C was quantitated after rapidly dissociating surface-bound insulin with an acidic buffer (pH 3.0). Of the 23 protease inhibitors tested, only chymotrypsin substrate analogues inhibited insulin internalization. Internalization was decreased 62-90% by five different chymotrypsin substrate analogues: N-acetyl-Tyr ethyl ester, N-acetyl-Phe ethyl ester, N-acetyl-Trp ethyl ester, benzoyl-Tyr ethyl ester, and benzoyl-Tyr amide. The effect of the substrate analogues in inhibiting insulin internalization was dose-dependent, reversible, and required the full structural complement of a chymotrypsin substrate analogue. Cell surface receptor number was unaltered at 12 degrees C. However, concomitant with their inhibition of insulin internalization at 37 degrees C, the chymotrypsin substrate analogues caused a marked increase (160-380%) in surface-bound insulin, indicating trapping of insulin-receptor complexes on the cell surface. Additionally, 1 mM N-acetyl-Tyr ethyl ester decreased overall insulin degradation by 15-20% and also prevented the chloroquine-mediated increase in intracellular insulin, further indicating that surface-bound insulin was prevented from reaching intracellular chloroquine-sensitive degradation sites. The internalization of insulin receptors that were photoaffinity labeled on the cell surface with B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin was also inhibited 70-90% by the five chymotrypsin substrate analogues, as determined by the effects of the analogues on the accumulation of trypsin-insensitive (intracellular) 440-kD intact labeled receptors. In summary, these results show that chymotrypsin substrate analogues efficiently inhibit the internalization of insulin and insulin receptors in adipocytes and implicate a possible role for endogenous chymotrypsin-like enzyme(s) or related substances in receptor-mediated endocytosis of insulin.     

Adenosine di- and triphosphate transport in mitochondria. Role of the amidine region for substrate binding and transport

A variety of base-modified nucleotide analogues was prepared and characterized as their alpha-32P- or U-14C-labeled compounds. Carrier-linked nucleotide binding and carrier-catalyzed exchange across the inner membrane of rat liver mitochondria were measured by using an inhibitor (atractyloside) stop method. Kinetic data of carrier-specific bound analogues were evaluated from Dixon plots and indicate that these analogues are competitive inhibitors for mitochondrial [14C]ADP uptake. Km and Vmax values for carrier-mediated uptake of nucleotide analogues were calculated from Lineweaver-Burk plots. By means of the analogues, a systematic mapping of the essential chemical and steric interactions between the transporter protein and the heterocycle of its substrate in the course of the binding as well as transfer step was achieved. Prerequisites for carrier-specific binding (recognition) are (A) an anti- or syn-positioned beta-glycosyl-linked heterocycle, (B) a nitrogen ring atom in position 7 for syn-structured analogues, and (C) an electron-rich region at the N(1) position, i.e., a permanent dipole moment oriented toward N(1) for anti-structured analogues. Additional requirements for subsequent transport catalysis are (A) a non-fixed anti-positioned base moiety with a beta-glycosyl torsion angle of about -20 degrees, (B) a C(6)-positioned amino group, and (C) an unsubstituted C(2) atom. The complementary binding site at the carrier protein to the N(1)-C(6)(-NH2) amidine region is proposed to be represented by two juxtaposed and invariant bonding points, i.e., an asparagine or glutamine residue.     

New agonist and antagonist analogues of bradykinin

Three new analogues of bradykinin (BK) have been tested for their agonistic and antagonistic actions on the rabbit jugular vein and the guinea pig ileum (B2 receptors), and six were studied on rabbit aorta strips (B1 receptors). Substitution of Gly4, Phe5, and Phe8 in BK with D-Trp gives analogues with a relative affinity lower than 1.0% as compared with BK. These analogues have no antagonistic properties on the rabbit jugular vein and on guinea pig ileum (B2 receptors). Substitution of Pro7 in des-Arg9-BK by Gly and by D-Ala give compounds that antagonise the effects of kinins on the rabbit aorta strips (B1-receptor system). These new antagonists are fairly potent with a pA2 value of 6.03 to 7.29 and seem competitive because the pA2--pA10 values approximate 0.95. These results suggest that the orientation of Phe8 is critical for the activation of B1 receptors by kinins.     

Stable-isotope probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating contaminated soil

[13C6]salicylate, [U-13C]naphthalene, and [U-13C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the "heavy" and "light" DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [13C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the 13C during the incubation did not appear to be an issue in this experiment.