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We describe our optimization efforts to improve the physicochemical properties, solubility, and off-target profile of 1, an inhibitor of TarO, an early stage enzyme in the biosynthetic pathway for wall teichoic acid (WTA) synthesis. Compound 1 displayed a TarO IC50 of 125 nM in an enzyme assay and possessed very high lipophilicity (clogP = 7.1) with no measurable solubility in PBS buffer. Structure–activity relationship (SAR) studies resulted in a series of compounds with improved lipophilic ligand efficiency (LLE) consistent with the reduction of clogP. From these efforts, analog 9 was selected for our initial in vivo study, which in combination with subefficacious dose of imipenem (IPM) robustly lowered the bacterial burden in a neutropenic Staphylococci murine infection model. Concurrent with our in vivo optimization effort using 9, we further improved LLE as exemplified by a much more druglike analog 26.

A series of benzimidazole analogs have been synthesized to improve the profile of the previous lead compounds tarocin B and 1. The syntheses, structure–activity relationships, and selected biochemical data of these analogs are described. The optimization efforts allowed the identification of 21, a fluoro-substituted benzimidazole, exhibiting potent TarO inhibitory activity and typical profile for a wall teichoic acid (WTA) biosynthesis inhibitor. Compound 21 displayed a potent synergistic and bactericidal effect in combination with imipenem against diverse methicillin-resistant Staphylococci.

High throughput screening for β-lactamase inhibitors afforded biphenyl hits such as 1. Hit confirmation and X-ray soaking experiments with Pseudomonas Aeruginosa AmpC enzyme led to the identification of an aryl boronic acid-serine complex 4, which was formed from phenyl boronic acid 8 (an impurity in compound 1) and ethylene glycol (the cryoprotectant in the soaking experiment).

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with β-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum β-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant Staphylococci.

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current β-lactam antibiotics and created an urgent need for new treatment options. We report an S. aureus phenotypic screening strategy involving chemical suppression of the growth inhibitory consequences of depleting late-stage wall teichoic acid biosynthesis. This enabled us to identify early-stage pathway-specific inhibitors of wall teichoic acid biosynthesis predicted to be chemically synergistic with β-lactams. We demonstrated by genetic and biochemical means that each of the new chemical series discovered, herein named tarocin A and tarocin B, inhibited the first step in wall teichoic acid biosynthesis (TarO). Tarocins do not have intrinsic bioactivity but rather demonstrated potent bactericidal synergy in combination with broad-spectrum β-lactam antibiotics against diverse clinical isolates of methicillin-resistant staphylococci as well as robust efficacy in a murine infection model of MRSA. Tarocins and other inhibitors of wall teichoic acid biosynthesis may provide a rational strategy to develop Gram-positive bactericidal β-lactam combination agents active against methicillin-resistant staphylococci.

Chopped basalt fiber reinforced wood sawdust panels were glued using melamine modified urea-formaldehyde resin. In order to improve the interfacial property, the basalt fibers was pre-treated by silane coupling agent. The tensile and bending strengths of the wood composites were tested, which indicated that the tensile and bending strengths of wood composites both reached their maximum values, 17.359MPa and 37.331MPa respectively, when the concentration of silane coupling agent is 0.75%.

Chopped basalt fibers untreated and treated by silane coupling agent were used to modify cement sample, in order to reduce water absorption. After fully soaked in water, the water content went up with the increase of fiber content. When the fiber content was 2.5Kg.m-3, the water absorption ratios of the samples reinforced by fibers without and with silane treatment were 4.83% and 5.44%, which were 57.1% and 39.5% less than the control group. This indicates that the fiber content was significant to water sorption and the influence of silane treatment is unnecessary for the reduction of water sorption.

The chopped basalt fibers and basalt glass powder were individually used as the flame retardant additives, and fir sawdust and melamine modified urea-formaldehyde resin together made the composite matrices. The fireproof wood particle panel was obtained by hot compressing the mixture, which may improve the inherent flammable defect of ordinary fiberboard. The limiting oxygen index indicated that the best flame retardant property of the basalt fiber reinforced wood composite was reached when the weight fraction of the chopped basalt fiber was 10%. With and without the basalt fiber additives, the difference of flame retardant performances was 10%. The wood composite, modified by basalt glass powder alone, improved its limiting oxygen index up to 11.7% when the weight fraction of the additives was 15%.

A series of pyrazoloquinolines, possessing (hetero)arylhydroxymethyl substituents at the quinoline C-4 position were evaluated as PDE10A inhibitors. Among these, methylpyrimidyl analogue 15 was identified as having good rodent and monkey exposure, and a MED of 10 mg/kg in an in vivo model.

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.

Bioassay-guided fractionation of an organic extract of the cell mass from a manipulated plant cell culture of Mirabilis jalapa led to the isolation and subsequent identification of three new phenolic compounds, 1, 2, and 3. The isoflavone 1 and dehydrorotenoid 2 were identified as the principal antifungal principles from this plant cell culture with IC(50)'s of 25 and 48 microg/mL, respectively, against the test organism, Candida albicans DSY1024. The rotenoid 3 was inactive at 200 microg/mL in this assay.

Bioassay-guided fractionation of an extract of a mixture of Microphilis guyanensis and Genipa americana collected in the rainforest of Suriname yielded the known alkaloid cryptolepine (2) as the major active compound in a yeast bioassay for potential DNA-damaging agents; the same compound was later reisolated from M. guyanensis. The structure of cryptolepine was identified unambiguously by spectral data and by its total synthesis. Several cryptolepine derivatives (3-29, 32-41) were synthesized based on modifications of the C-2, N-5, N-10, and C-11 positions. Two cryptolepine dimers (30, 31) were also prepared. The structure modifications did not result in compounds with a higher potency than the parent compound cryptolepine in the yeast assay system, although some derivatives did show significant activity. Selected compounds (6, 7, 17, 22, 23, 26, and 27) were also tested for cytotoxicity in mammalian cell culture, and two compounds showed significant cytotoxic activity.

The cytotoxic potential and structure activity relationships of several indole metabolites obtained from feeding experiments using Streptomyces staurosporeus, a staurosporine producer, are described. 6-Fluoro-tryptamine and N b -acetyl-tryptamine-5- O -a-L-rhamnopyranoside, a metabolite obtained from the feeding experiment, demonstrated greatest activity with KB cells in culture, by means of an unknown mechanism.

A 1:1 mixture of two epimers of an unusual linear diterpene, lingulatusin, was isolated from the whole plant of Aster lingulatus . The structure was determined on the basis of IR, MS and extensive NMR spectral studies, and X-ray crystallographic data.

Bioassay-guided fractionation of Eschweilera coriacea collected in the lowland wet forest of Suriname yielded the new but only weakly active ellagic acid derivative eschweilenol A (1) and the two new but inactive ellagic acid derivatives eschweilenol B (2) and eschweilenol C (3). The four known compounds, sucrose, ellagic acid, 3-O-galloylepigallocatechin, and epigallocatechin, were also isolated. The structures of the three new compounds were determined by spectrometric methods, primarily from the HMQC, HMBC, NOESY, and ROESY NMR techniques, and chemical methods, including methylation and triethylsilylation. The location of a hydroxyl group in one ellagic acid derivative was determined by a new technique involving an NOE correlation of the protons of a triethylsilyl derivative with a proton on a neighboring aromatic ring.

The roots of Ononis spinosa subsp. leiosperma (Leguminosae) afforded a new glycoside, spinonin (1), possessing a novel skeleton, in addition to the known isoflavonoid glycoside, ononin [7beta-(glucosyloxy)formononetin] (2) and the known pterocarpan, 7-demethoxy-7-D-(glucosyloxy)homopterocarpin (3). The structure of the new isolate was elucidated by spectral methods including 1H and 13C NMR, COSY, APT, HETCOR, HMBC, NOESY, CD, FABMS, HRMS, EIMS, CIMS, and some chemical reactions. Spinonin was inactive against a number of human cancer cell lines and HIV-1 reverse transcriptase. The compounds 1 and 3 showed weak activity against Pseudomonas aeruginosa, whereas 2 was active against beta-hemolytic Streptococcus.

Two tetracyclic diterpene polyesters, paralinones A and B, with a new carbon framework, were isolated from an acetone extract of the whole plant of Euphorbia paralias. The structures of the compounds were elucidated by high field spectroscopic methods, including 2D NMR techniques and X-ray crystallography.

Two ergostane related steroids, zhankuic acids D and E together with three lanosta related triterpenes, 15α-acetyl-dehydrosulphurenic acid, dehydroeburicoic acid, dehydrosulphurenic acid were isolated from the fruit body of the fungus Antrodia cinnamomea. Their structures were determined by spectral analyses and comparison with known compounds.

Heavy metals, including zinc (Zn) and cadmium (Cd), are potentially important genotoxic agents in our environment. Here we report that human DNA ligase I, the major form of the enzyme in replicative cells, is a target for Zn and Cd ions. ZnCl2 at 0.8 mM caused complete inhibition of DNA ligase I activity, whereas only 0.04 mM CdCl2 was required to achieve a similar effect. Both metals affected all three steps of the reaction, namely, the formation of ligase-AMP intermediate, the transfer of the AMP to DNA and the ligation reaction that succeeds the formation of the AMP-DNA complex. Unlike F-ara-ATP and the natural protein inhibitor of DNA ligase-I, these metals may affect different domains of the enzyme. Moreover, these metal ions did not increase the rate of misligation of F-ara-A-modified DNA or mismatched DNA substrates, but considerable misligation was observed for the T:C mispairing. These data support the notion of high fidelity of the human DNA ligases and that the major action of these metal ions on the enzyme is their inhibitory function.

Chloroform-soluble extracts of the stems and of the mixed stems and stem bark of Lophopetalum wallichii were found to be inhibitory in a farnesyl protein transferase (FPTase) bioassay system. During the course of activity-guided fractionation, the known lupane-type triterpenes, ochraceolide A (1), ochraceolide B (2), betulin, and lupeol and the new lupane lactone, dihydro ochraceolide A (4), were isolated. The stereochemistry of the epoxide group of ochraceolide B (2) was determined by preparation of both epoxide isomers [2, and the new semisynthetic derivative, 20-epi-ochraceolide B (3)] from 1. The structure of 4 was established by reduction of 1 with sodium borohydride. Compounds 1 and 2 exhibited significant inhibitory activity in the FPTase assay (IC50 values of 1.0 and 0.7 microgram/mL, respectively). Lupeol was found to be weakly active (IC50 65.0 micrograms/mL) in this test system, whereas no significant inhibition was detected for betulin or compounds 3 or 4. When evaluated against a panel of human cancer cells in culture, compounds 1 and 4 were modestly cytotoxic. Compounds 2 and 3 were not active in the panel.

Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact > 25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.

DNA ligation catalyzed by all DNA ligases involves two intermediary steps, the formation of the ligase-AMP and the AMP-DNA complexes. A method was developed to purify and analyze the AMP-DNA intermediate from the DNA ligation reaction catalyzed by DNA ligases. This AMP-DNA complex was maximally accumulated by preincubation of human DNA ligase I or II with ATP, followed by interaction with the DNA substrate for 5 s at 0 degrees C. The gel-purified AMP-DNA complex maintained its property as a ligation intermediate. The AMP was directly linked to the 5'-phosphate of DNA with a pyrophosphate bond. The successive ligation reaction following the AMP-DNA complex formation required DNA ligase and Mg2+ ion but was inhibited by ATP and pyridoxal 5'-phosphate, indicating that the availability of the AMP binding site in the enzyme is essential for the completion of the reaction. Furthermore, the formation of the AMP-DNA complex and the subsequent DNA ligation were substrate specific for human DNA ligases I and II. These data, together with previously reported results, suggest that a major difference between human DNA ligases I and II is in their DNA-binding domains. The methods make it convenient to study in depth the kinetics of the overall DNA ligation.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (F-ara-A) is an analogue of adenosine and deoxyadenosine with potent anti-tumor activity. The mechanism of action for this compound has been elucidated as the inhibition of DNA and RNA synthesis, induction of DNA fragmentation, and genetic damage. This study demonstrated that DNA ligase I, an enzyme involved in DNA replication, is a target for the drug action. F-ara-adenine triphosphate (F-ara-ATP) at 80 microM inhibited the activity of DNA ligase I by more than 90%. In contrast, eight other related nucleoside analogues showed no effect on the enzyme activity at 200 microM. F-ara-ATP inhibited DNA ligation in two distinct ways. First, F-ara-ATP directly interacted with DNA ligase I and inhibited the formation of the ligase-AMP complex. This inhibition could not be reversed when free F-ara-ATP was eliminated from the treated enzyme; however, the addition of pyrophosphate, followed by gel filtration chromatography, restored enzyme activity, indicating that F-ara-ATP bound to the enzyme and altered the AMP-binding site. Secondly, the activity of DNA ligase I was inhibited when F-ara-ATP was incorporated into the 3' terminus of the DNA substrate. The dual mode of inhibition of DNA ligase I by F-ara-ATP indicates that its effect on DNA ligation may be important in the inhibition of DNA synthesis and the cytotoxicity of F-ara-A.

Four new caryophyllene derivatives, Sch 725432 (1), Sch 601253 (2), Sch 601254 (3), and Sch 725434 (4), were isolated from the fungal fermentation broth of Chrysosporium pilosum by reversed-phase HPLC purification. The structure elucidation of trioxygenated caryophyllenes 1−4 was accomplished on the basis of spectroscopic data interpretation. Sch 725434 (4) possesses a dihydrofuran-3-one ring, forming a tricyclic ring skeleton, which represents an unprecedented ring skeleton for the caryophyllene-type of sesquiterpenes. Compounds 1−4 were evaluated for their antifungal activity.

The feeding of (13)C- and (2)H-enriched methionine to Streptomyces staurosporeus established that the methyl carbon and proton source of both the 3'-O- and 4'-N-methyl groups of staurosporine (1) was methionine and that all three methyl protons from methionine were retained on 1. In the presence of the methyltransferase inhibitor, sinefungin, the biosynthesis of staurosporine was blocked at the last step, O-methylation. An intermediate, 3'-demethoxy-3'-hydroxystaurosporine (2), was efficiently accumulated in the medium. Other general methyltransferase inhibitors failed to produce any other staurosporine intermediates or analogues.

The fate of the nitrogen atoms in tryptophan during biosynthesis of the antitumor antibiotic staurosporine (1), a potent PKC inhibitor, has been studied using [ 15 N 2 ]-L-tryptophan and 15 NH 4 NO 2 . The results indicate that the nitrogen atom on the side chain of tryptophan is cleaved during staurosporine synthesis.

Zhankuic acid F (1), a new steroid acid, was isolated from the fruit bodies of Antrodia cinnamomea Chang & Chou, sp. nov. (Polyporaceae). The structure of 1 was elucidated by detailed analysis of 1D- and 2D-NMR spectra. Compound 1 appeared as a major product from the microbial transformation (Mucor racemosus) of zhankuic acid A (2).

Bioassay-guided fractionation of an active fraction from a marine sponge Topsentia sp. in our marine fraction library (MFL) led to the isolation and identification of halistanol sulfate ( 1 ) and a new sterol sulfate Sch 572423 ( 2 ). Compounds 1 and 2 were identified as P2Y 12 inhibitors with IC 50 of 0.48 and 2.2 μM, respectively. The general method of purification for the MFL library and the structure elucidation of compound 2 are described.

Texto completo: acesso restrito. p.443–447 Submitted by Suelen Reis (suelen_suzane@hotmail.com) on 2013-01-22T12:31:44Z No. of bitstreams: 1 David.pdf: 451807 bytes, checksum: 5500fa37d59eea96412da5a91540f483 (MD5) Made available in DSpace on 2013-01-22T12:31:44Z (GMT). No. of bitstreams: 1 David.pdf: 451807 bytes, checksum: 5500fa37d59eea96412da5a91540f483 (MD5) Previous issue date: 1999 A new bis-diterpene, moldenin (6), derived from the esterification of 3-oxo-8(17)-15-labdenic acid with 3β-hydroxy-8(17)-15-labdenol, four normal-labdane diterpenes 1–4, and betulinic acid were isolated from the stems of Moldenhawera nutans. The structures were established on the basis of their spectral data.

During our continuing studies of staurosporine (1) biosynthesis and the metabolism of indole derivatives, three additional novel metabolites, beta-hydroxy-Nb-acetyltryptamine (7) and its 5- and 6-fluoro derivatives (8 and 9, respectively), were isolated, respectively, from the feeding of tryptamine and 5- and 6-fluorotryptamines using Streptomyces staurosporeus. In addition, two new 5-hydroxytryptamine (serotonin) metabolites, Nb-acetyltryptamine-5-O-beta-D-quinovopyranoside (11) and Nb-acetyltryptamine-5-O-alpha-L-rhamnopyranoside (12), were isolated along with 5-hydroxy-Nb-acetyltryptamine (10). From a glucose containing medium, 6-O-acetylglucose and its 13C uniformly labeled derivative were isolated from a culture of S. staurosporeus during biosynthetic studies of 1. In 2-deoxyglucose metabolism studies, 6-O-acetyl-2-deoxyglucose was isolated and identified as a metabolic product of 2-deoxyglucose. A copper-enzyme inhibitor, N,N-diethyldithiocarbamic acid, was evaluated for the accumulation of staurosporine intermediates; however, no modified staurosporines were observed.

Texto completo: acesso restrito. p.967–968 Submitted by Suelen Reis (suelen_suzane@hotmail.com) on 2013-01-31T12:49:43Z No. of bitstreams: 1 Chavez.pdf: 207330 bytes, checksum: 13e2fbad59c0e189f97fd52a5ed73fd3 (MD5) Made available in DSpace on 2013-01-31T12:49:43Z (GMT). No. of bitstreams: 1 Chavez.pdf: 207330 bytes, checksum: 13e2fbad59c0e189f97fd52a5ed73fd3 (MD5) Previous issue date: 1997 Leaves of Acanthosyris paulo-alvinii afforded the new alkaloid 2,3-methylenedioxy-4,7,8-trimethoxy-quinoline (1), as well as the known compounds loliolide (2) and 3-hydroxy-5,6-epoxy-β-ionone (3). 13C NMR spectroscopic assignments are reported.

From a tryptophan metabolic study, 3-(hydroxyacetyl)indole, indole-3-carboxaldehyde, and o-aminobenzoic acid were obtained as tryptophan metabolites from a staurosporine (1) producer, Streptomyces staurosporeus. A new tryptamine metabolite, (3aR,8aS)-1-acetyl-1,2,3,3a,8,8a-hexahydropyrrolo[2,3- b]indol-3a-ol (2), was isolated along with Nb-acetyltryptamine using S. staurosporeus fed with tryptamine. The unusual compound 2 was proposed as a further metabolite of Nb-acetyltryptamine through an enzymatic oxidative cyclization. Examination of the metabolites from the feeding of 5- and 6-fluorotryptamines using the same microorganism afforded the two novel compounds 3 and 4 as the 5- and 6-fluoro derivatives of 2. However, 5-hydroxytryptamine failed to generate the 5-hydroxy derivative of 2. Indole-ring-substituted metabolites of staurosporine (1) were not observed.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.

Continuation of a previous study on Renealmia alpinia resulted in the isolation of the new labdane diterpenoid 3, together with two known diterpenoids. The structure of the new diterpenoid was determined by a combination of NMR techniques and HRFABMS.

Integration host factor (IHF) is a small heterodimeric protein that specifically binds to DNA and functions as an architectural factor in many cellular processes in prokaryotes. Here, we report the crystal structure of IHF complexed with 35 bp of DNA. The DNA is wrapped around the protein and bent by >160°, thus reversing the direction of the helix axis within a very short distance. Much of the bending occurs at two large kinks where the base stacking is interrupted by intercalation of a proline residue. IHF contacts the DNA exclusively via the phosphodiester backbone and the minor groove and relies heavily on indirect readout to recognize its binding sequence. One such readout involves a six-base A tract, providing evidence for the importance of a narrow minor groove.