Identification and characterization of novel antimicrobial decapeptides generated by combinatorial chemistry

Novel combinatorial libraries consisting of simplified amino acid sequences were designed to screen for peptides active against the Candida albicans membrane. A novel decapeptide, KKVVFKVKFK, that had a unique primary amino acid sequence was identified in this work. This peptide irreversibly inhibited the growth of C. albicans and showed a broad range of antibacterial activity but no hemolytic activity. Circular dichroism spectra revealed that the predominant secondary structure of this peptide strongly depended on the membrane-mimetic environments; the peptide preferred to form an amphipathic alpha-helical structure in the presence of 50% trifluoroethanol, while it preferred to adopt a distorted alpha-helical structure in the presence of sodium dodecyl sulfate micelles. Experiments in which dye was released from vesicles indicated that this novel antimicrobial peptide killed microorganisms through the action on the membrane as its primary target. Replacement of amino acids in this active decapeptide on the basis of information from the libraries could provide unique information about factors affecting its antimicrobial activity such as its secondary structure, net positive charge, and hydrophobicity.     

Discovery of a novel tetrahydroacridine acetylcholinesterase inhibitor through an indexed combinatorial library

BACKGROUND: Methods for the rapid and efficient preparation of drug candidates through combinatorial chemistry are of increasing interest. We have previously reported an indexed combinatorial library method that allows both the preparation and testing of compounds in solution. We set out to apply this method to develop more effective analogs of the known, marketed drug tacrine, an acetylcholinesterase inhibitor. RESULTS: A one-step condensation of cyclohexanones with cyanoanilines to generate tetrahydroacridine pools was developed. The resulting library of (formally) 72 tetrahydroacridines was screened against acetylcholinesterase, and a compound 10-fold more potent than tacrine, 7-nitrotacrine, was discovered. Its increased potency could be readily explained by examining the known structure of the complex of acetylcholinesterase with tetrahydroacridine. CONCLUSIONS: In this work, we have provided a relatively rare example of carbon-carbon bond formation in a pool synthesis and have discovered a potentially useful acetylcholinesterase inhibitor.     

Discovery of IRL 3461: a novel and potent endothelin antagonist with balanced ETA/ETB affinity

IRL 3461, N-butanesulfonyl-[N-(3,5-dimethylbenzoyl)-N-methyl-3-[4-(5-+ ++isoxazolyl) -phenyl]-alanyl]-(L)-valineamide, a potent and bifunctional (ETA + ETB) [Ki(ETA) = 1.8 nM, Ki(ETB) = 1.2 nM] antagonist was discovered by structural modification of IRL 2500, an ETB selective antagonist. IRL 3461 was found to be stable on incubation with human, rat, mouse, and guinea pig plasmas.     

Discovery of a new cyclooxygenase-2 lead compound through 3-D database searching and combinatorial chemistry

Prolonged nerve conduction blockade has been proposed to result from the summed effects of charged and neutral local anaesthetics. Thirty-seven patients were randomly allocated to receive intravenous patient-controlled analgesia alone or combined with intercostal blockade (T7-T11) with a mixture of 0.45% bupivacaine and 0.6% phenol for post-cholecystectomy analgesia. Adequacy of pain relief was measured by patient scores on a 10-cm visual analogue scale and by dose-demand ratio, amounts of loading dose and total consumption of morphine and also the duration of patient-controlled analgesia in each group. No differences were found between groups in post-operative scores, dose-demand ratios and loading doses of morphine. However, in the combined treatment group, a significantly lower total consumption of morphine (P < 0.05), associated with a shorter duration of patient-controlled analgesia (P < 0.02) and a decreased mean number of unsuccessful demands (P < 0.001) were recorded. Intercostal blockade with bupivacaine-phenol supplements intravenous patient-controlled analgesia for post-cholecystectomy pain relief.     

New phthalimide derivatives with potent analgesic activity: II

Seven new phthalimide derivatives (9a-g) with 1,2,4-oxadiazol-5-yl methyl group attached to nitrogen have been synthesized from N-phthaloylglycine 6 and arylamidoximes 7a-g. All of these showed potent analgesic effect with acetic acid induced "writhing" test in mice. One of the better compounds (ID50 = 2.2 mg/Kg i.p.) has been found to be 9a which also demonstrates analgesic activity against inflammatory pain.     

Behavioral effects of HT-90B, a putative novel anxiolytic agent with potent antidepressive activity

1. HT-90B,((-)-N-[2-(8-methyl-1,4-benzodioxane-2-ylmethyl)am in o]ethyl]tricyclo[3,3,1,1(3,7)] decane-1-carboxamide), is a novel serotonin 5-HT(1A) full agonist with serotonin 5-HT(2) antagonistic action. 2. In a water licking conflict test, HT-90B (3-30 mg/kg, po) was more effective than buspirone in abolishing response suppression. 3. In a forced swim test, HT-90B (3-30 mg/kg, po) reduced the duration of immobility after a single oral dose, which contrasts with the requirement of multiple doses for desipramine (as a standard antidepressant) to produce the effect. 4. These two behavioral effects of HT-90B (20 mg/kg,po) were still evident even 4 hr after oral administration. And no tolerance was observed for these behavioral effects after repeated oral administration (10 mg/kg, b.i.d., 2 weeks). 5. HT-90B hardly produced any diazepam- or buspirone-like side effects. 6. In conclusion, it is suggested HT-90B is a potent anti-depressive anxiolytic with minimal side effects.     

Structures of novel acidic galactooligosaccharides synthesized by Bacillus circulans beta-galactosidase

The structures of acidic oligosaccharides synthesized by a transglycosylation reaction by Bacillus circulans beta-galactosidase, using lactose as the galactosyl donor, and N-acetylneuraminic acid (NeuAc) and glucuronic acid (GlcUA) as the acceptors were investigated. Acidic oligosaccharides thus synthesized were purified by anion exchange chromatography and charcoal chromatography. The MS and NMR studies indicated that the acidic oligosaccharides from NeuAc were Gal beta-(1-->8)-NeuAc, Gal beta-(1-->9)-NeuAc, and Gal beta-(1-->3)-Gal beta-(1-->8)-NeuAc, and those from GlcUA were Gal beta-(1-->3)-GlcUA and Gal beta-(1-->4)-Gal beta-(1-->3)-GlcUA. These are novel acidic galactooligosaccharides.     

Encoded combinatorial chemistry: synthesis and screening of a library of highly functionalized pyrrolidines

The application of a new encoding technology for drug discovery is described. A combinatorial library of mercaptoacyl pyrrolidines has been prepared on a beaded polymeric support. Each polymer bead carries one library constituent in association with an oligomeric "tag," the structure of which is a record of the specific reagents from which that library member was prepared. After the ligands were solubilized, an array of such beads was screened for angiotensin-converting enzyme inhibitory activity, and the structures of active pyrrolidines were deduced by analysis of the associated tags at sub-picomole levels. Several extremely potent enzyme inhibitors were identified, many from multiple beads. The most potent inhibitor was found to have a Ki of 160 pM, approximately 3-fold more active than captopril in the same assay. Direct comparison with iterative deconvolution shows that the encoded screening strategy is a much more efficient means for extracting information from such compound collections, producing more data on a larger number of active structures.     

In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution

BACKGROUND: It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites. RESULTS: We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA 'lectins' that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection. CONCLUSIONS: Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these 'low molecular-weight effectors' may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.     

Herbal mixtures with claimed slimming activity: determination by TLC and HPLC of illegally added drugs

A method for the detection and quantitation of several undeclared drugs in herbal preparations with slimming activity is proposed. Samples containing various anorexics, hypoglycemics and antidepressants were prepared by addition of the drugs to a synthetic mixture containing the most commonly used plant powders for those preparations. Each sample was subjected to a treatment that permitted, after a simple ethanolic extraction, the identification of the drugs by TLC using three different solvent systems. A further purification of the ethanolic solution through a polyamide column allowed for quantitative analysis of the drugs by a RP-HPLC method. The analytical recovery was good (88-97%); the calibration curves were linear over a wide range of drug concentrations (30-500 micrograms/ml) (r > 0.9995); the precision was high (CV% = 0.4-2.8) as well as the accuracy (96-102%).     

Enhanced photocatalytic activity of Ce-doped ZnO nanopowders synthesized by combustion method

Facile and fast combustion method was used to synthesize Zn O and Ce-doped Zn O(CZO) nanocrystalline powders photocatalysts with different cerium concentrations(0.5 wt.%–10.0 wt.%) followed by calcination at 700 oC for 3 h. The prepared samples were characterized by a variety of characterization techniques such as X-ray diffraction(XRD), scanning electron microscopy(SEM) combined with energy dispersive X-ray diffraction(EDX), transmission electron microscopy(TEM), UV-visible spectroscopy, BET surface area analyser and photoluminescence spectroscopy(PL), to study the crystal structure, surface morphology, chemical composition and optical properties. It was observed from XRD results that synthesized powders had hexagonal wurtzite structure with the smallest crystallite size about 13 nm. Absorption spectra showed that cerium doping enhanced the light absorption properties towards the visible light region. Photoluminescence spectra for Ce-doped Zn O samples exhibited relatively weak near band edge(NBE) emission peaks as compared to that of pristine Zn O. The photocatalytic activities of the prepared samples were evaluated by photocatalytic degradation of Rhodamine B(Rh B) under UV light and visible light(λ≥420 nm) irradiation. The textile mill effluent containing organic matters was also treated under sunlight using photocatalysis and the reduction in the chemical oxygen demand(COD) of the treated effluent revealed a complete destruction of the organic molecules along with colour removal. The results showed that the CZO photocatalyst doped with 3.0 wt.% cerium exhibited four times enhanced photocatalytic activity compared to pure Zn O. The enhanced photocatalytic activity could be attributed to extended visible light absorption and inhibition of the electron-hole pair’s recombination.     

Enhancement of antimicrobial activity of neuropeptide Y by N-terminal truncation

The activity of neuropeptide Y (NPY) against Candida albicans, which was revealed to be fungicidal, was enhanced significantly by the truncation of amino acid residues at the N terminus. The most active peptides (MICs, approximately 1 microM) were about 10-fold more potent than the intact NPY (MIC, approximately 10 microM). The enhancement was weakened by the replacement of the N terminus by negatively charged residues and/or acylation of the alpha-amino group. These results suggest that only the alpha-helical region of NPY is necessary for the antimicrobial activity and that the net charge of the peptide is important for the activity.     

An antimicrobial activity of cytolytic T cells mediated by granulysin

Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.     

Discovery of a novel series of potent and selective substrate-based inhibitors of p60c-src protein tyrosine kinase: conformational and topographical constraints in peptide design

On the basis of the efficient substrate for p60c-src protein tyrosine kinase (PTK) YIYGSFK-NH2 (1) (Km = 55 microM) obtained by combinatorial methods, we have designed and synthesized a series of conformationally and topographically constrained substrate-based peptide inhibitors of this enzyme, which showed IC50 values in the low-micromolar range (1-3 microM). A "rotamer scan" was performed by introducing the four stereoisomers of beta-Me(2')Nal in the postulated interaction site of the peptide inhibitor 23(IC50 = 1.6 microM). This substitution led to selective and potent inhibitors of p60c-src PTK; however, no substantial difference in potency was observed among them. This and the results of the "stereochemical scan" performed at residues 2 and 7 of 3 (peptides 19-21), which form the disulfide bond, may suggest that the enzyme active site does not have rigid topographic requirements and thus is able to achieve important conformational changes to bind the ligand as long as the pharmacophore pattern in the inhibitor is conserved. Two new potent iodo-containing nonphosphorylatable tyrosine analogues were also incorporated into our lead inhibitory sequence 23, producing the most potent inhibitors for p60c-src PTK identified thus far in our studies. Compounds 29 and 30 exhibit IC50 values of 0.13 and 0.54 microM, respectively. Peptide 29 is 420-fold more potent than the parent peptide 1. Selectivity studies of peptides 23-30 toward p60c-src, Lyn, and Lck PTK showed in general high Lyn/Src and moderate Lck/Src selectivity ratios. We found that the chi1 space constraints of the specialized amino acids, introduced at position 3 of the peptide lead 23, were not as important as the configuration of the Calpha of that residue to recognize the subtle chemical environment surrounding the active site of Src and Lck PTK, as reflected on the obtained Lck/Src selectivity ratios.     

Aspects of the interf Ci l chemistry of nickel extraction with LIX63-HDNNS mixtures

The extraction of nickel from acidic aqueous solutions with LDC63(HOx:)-dinonylnaphtha-lenesulfonic acid (HDNNS) mixtures has been studied using purified reagents. Interfacial tension data and infrared spectra are provided which indicate the presence of an oximesulfonate interaction. Extraction of nickel is significant only if HDNNS micelles and HO* molecules are present together in the organic phase. The stoichiometry of the extracted nickel complex appears to be Ni: oxime = 1:3. An extraction mechanism is proposed involving HDNNS micellar enhancement of the oxime-nickel chelation reaction.     

Small peptide libraries: combinatorial split-mix synthesis followed by combinatorial amino acid analysis of selected variants

Site-related categories of suicide enable certain characteristics to be worked out which speak both for, as well as against, a voluntary decision, and can generally be viewed as indicative. However, taking other site-unrelated factors into account, there are necessarily differences which can only be conditional to the particular case in question.     

4-(3'-Bromo-4'hydroxylphenyl)-amino-6,7-dimethoxyquinazoline: a novel quinazoline derivative with potent cytotoxic activity against human glioblastoma cells

The novel quinazoline derivative 4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154) exhibited significant cytotoxicity against U373 and U87 human glioblastoma cell lines, causing apoptotic cell death at micromolar concentrations. The in vitro antiglioblastoma activity of WHI-P154 was amplified > 200-fold and rendered selective by conjugation to recombinant human epidermal growth factor (EGF). The EGF-P154 conjugate was able to bind to and enter target glioblastoma cells within 10-30 min via receptor (R)-mediated endocytosis by inducing internalization of the EGF-R molecules. In vitro treatment with EGF-P154 resulted in killing of glioblastoma cells at nanomolar concentrations with an IC50 of 813 +/- 139 nM, whereas no cytotoxicity against EGF-R-negative leukemia cells was observed, even at concentrations as high as 100 microM. The in vivo administration of EGF-P154 resulted in delayed tumor progression and improved tumor-free survival in a severe combined immunodeficient mouse glioblastoma xenograft model. Whereas none of the control mice remained alive tumor-free beyond 33 days (median tumor-free survival, 19 days) and all control mice had tumors that rapidly progressed to reach an average size of > 500 mm3 by 58 days, 40% of mice treated for 10 consecutive days with 1 mg/kg/day EGF-P154 remained alive and free of detectable tumors for more than 58 days with a median tumor-free survival of 40 days. The tumors developing in the remaining 60% of the mice never reached a size > 50 mm3. Thus, targeting WHI-P154 to the EGF-R may be useful in the treatment of glioblastoma multiforme.