Structure, histone deacetylase, and antiprotozoal activities of apicidins B and C, congeners of apicidin with proline and valine substitutions.

[structure: see text]. Isolation and structure elucidation of two novel cyclic tetrapeptides that show a variety of potent antiprotozoal activities by reversibly inhibiting HDAC have been reported. These are the new members of a unique family of cyclic tetrapeptides that do not require the electrophilic alpha-epoxyketone moiety of HC-toxin, trapoxin A, or chlamydocin for their potent activities against HDAC and the malarial parasite.

Solvation of acylium fragment ions in electrospray ionization quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry.

In electrospray ionization (ESI) quadrupole ion trap and Fourier transform ion cyclotron resonance mass spectrometry, certain fragment ions (e.g. acylium ions) generated either during the ion transportation process (in the source interface region) or in the ion trap are found to undergo ion--molecule reactions with ESI solvent molecules (water, acetonitrile and aliphatic alcohols) to form adduct species. These unexpected solvated fragment ions severely complicate the interpretation of mass spectrometic data. High-resolution accurate mass measurements are important in establishing the elemental compositions of these adduct species and preventing erroneous data interpretation.

Hyalodendrosides A and B, antifungal triterpenoid glycosides from a lignicolous hyphomycete, Hyalodendron species.

Two antifungal triterpenoid glycosides, hyalodendrosides A and B (1 and 2), were isolated from a solid matrix fermentation of a lignicolous hyphomycete, Hyalodendron sp. Their structures were determined based upon extensive examination of spectral parameters, particularly NMR and MS data. Both compounds have beta-linked glucose moieties. Compounds 1 and 2 show weak to moderate antifungal activity against some clinically relevant fungi.

The design and development of an integrated natural products screening database.

We designed and developed NEXUS--a new natural products screening database and related suite of software applications--to utilize the spectacular increases in assay capacity of the modern high throughput screening (HTS) environment. NEXUS not only supports seamless integration with separate HTS systems, but supports user-customized integration with external laboratory automation, particularly sample preparation systems. Designed and developed based on a detailed process model for natural products drug discovery, NEXUS comprises two integrated parts: (1) a single schema of Oracle tables and callable procedures and functions, and (2) software "front-ends" to the database developed using Microsoft Excel and Oracle Discovery/2000. Many of the back-end processing functions were written in Programming Language/Structured Query Language (PL/SQL) to provide an Application Programmer's Interface, which allows end users to create custom applications with little input from information technology professionals.

Quinoxapeptins: novel chromodepsipeptide inhibitors of HIV-1 and HIV-2 reverse transcriptase. I. The producing organism and biological activity.

Quinoxapeptin A and B are novel chromodepsipeptides which were isolated from a nocardioform actinomycete with indeterminant morphology. Quinoxapeptins A and B are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase and almost equally active against two single mutants forms as well as a double mutant form of HIV-1 reverse transcriptase. Quinoxapeptin A and B are specific inhibitors of HIV-1 and HIV-2 reverse transcriptase because they did not inhibit human DNA polymerase alpha, beta, gamma and delta. Quinoxapeptin A and B are structurally similar to luzopeptin A which was also active against HIV-1 and HIV-2 reverse transcriptase.

L-735,334, a novel sesquiterpenoid potassium channel-agonist from Trichoderma virens.

A novel oleic acid ester of the carotane sesquiterpene 14-hydroxy CAF-603 was isolated from Trichoderma virens grown in a solid brown rice-based medium, a solid millet-based medium, or a mannitol-based liquid medium. Its structure was determined on the basis of ms and nmr analysis. It retains distinct biological activity on the high conductance calcium-activated potassium channel, unlike its analogues 14-hydroxy CAF-603, CAF-603 3-oleate, or CAF-603 3-linoleate.

Development of fibroblast-seeded ligament analogs for ACL reconstruction.

We fabricated "ligament analogs" in vitro by seeding high-strength resorbable collagen fiber scaffolds with intraarticular (anterior cruciate ligament, ACL) or extraarticular (patellar tendon, PT) rabbit fibroblasts. Fibroblasts attached, proliferated, and secreted new collagen on the ligament analogs in vitro. Fibroblast function depended on the tissue culture substrate (ligament analog vs. tissue culture plate) and the origin of the fibroblasts (ACL vs. PT) PT fibroblasts proliferated more rapidly than ACL fibroblasts when cultured on ligament analogs. Collagen synthesis by ACL and PT fibroblasts was approximately tenfold greater on ligament analogs than on tissue culture plates. The composition, structure, and geometry of the collagen fiber scaffolds may promote collagen synthesis within ligament analogs in vitro. Ligament analogs roughly approximate the structure and strength of native ligament tissue. Ongoing in vivo studies suggest that autogenous fibroblast-seeded ligament analogs remain viable after implantation into the knee joint. With further development, ligament analogs may be useful as implants for ACL reconstruction surgery.

Mechanism of inhibition of human leucocyte elastase by beta-lactams. 2. Stability, reactivation kinetics, and products of beta-lactam-derived E-I complexes.

The monocyclic beta-lactams reported by Knight et al. [Knight, W. B., et al. (1992) Biochemistry 31, 8160; Chabin, R., et al. (1993) Biochemistry 32, 8970] as inhibitors of human leucocyte elastase (HLE) produce stable HLE-inhibitor complexes that slowly reactivate with half-lives ranging from less than 1 to 15 h at 37 degrees C. The complexes produced between PPE and two C-3 dimethyl-substituted beta-lactams are less stable than those produced between HLE and analogous C-3 diethyl-substituted lactams. The stability of the HLE-I complexes is governed primarily by the structure of the substituted urea portion of the inhibitors and not by the identity or presence of a leaving group at C-4 of the lactam ring. In some cases substitutions on the urea portion of the inhibitors yielded complexes that displayed biphasic reactivation kinetics. This suggests the presence of at least two different complexes. The stereochemistry of the leaving group at C-4 has a small effect on the stability of the final complex (1.3-2-fold); therefore, the identity of the final complex is dependent upon the initial stereochemistry at that position. The stability of the complexes was relatively insensitive to hydroxylamine, which suggests that the acyl-enzymes are protected from nucleophilic "rescue". The rate of reactivation of the complex derived from L-680,833,[S-R*,S*)]-4-[(1-(((1-(4- methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-oxo-4-azetidinyl)ben zeneacetic acid, was pH independent, while the L-684,481, (R)-(1-(((1-(4-methylphenyl)butyl)amino)carbonyl)-3,3-diethyl-2-azeti din one generated complex displayed a pH-dependent reactivation rate. In the latter case, the increase in reactivation rate with pH displayed a pKa of 7.2. This is consistent with the requirement for base catalysis by the active site histidine to regenerate enzymatic activity. Reactivation of the L-680,833-derived complex produced different products as a function of pH, suggesting two different pH-dependent routes of reactivation. At low pH a route that produced primarily the substituted urea is favored, while at higher pH production of two six-membered ring diastereomers competes with urea generation. Thus, the apparent pH independence of the return of activity is the result of two offsetting pathways. Other compounds such as L-670,258, (S)-4-[((((2-naphthylmethyl)amino)carbonyl)-3,3-diethyl-4-oxo-2- azetidinyl)oxy]benzoic acid, reactivate by these two routes as well as by aminolysis by the other urea nitrogen to produce an additional regioisomer. The temperature dependence of the reactivation of the complexes derived from L-684,481 and L-680,833 suggests different mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action.

Tubulin polymerization into microtubules is a dynamic process, with the equilibrium between growth and shrinkage being essential for many cellular processes. The antineoplastic agent taxol hyperstabilizes polymerized microtubules, leading to mitotic arrest and cytotoxicity in proliferating cells. Using a sensitive filtration-calorimetric assay to detect microtubule nucleating activity, we have identified epothilones A and B as compounds that possess all the biological effects of taxol both in vitro and in cultured cells. The epothilones are equipotent and exhibit kinetics similar to taxol in inducing tubulin polymerization into microtubules in vitro (filtration, light scattering, sedimentation, and electron microscopy) and in producing enhanced microtubule stability and bundling in cultured cells. Furthermore, these 16-membered macrolides are competitive inhibitors of [3H]taxol binding, exhibiting a 50% inhibitory concentration almost identical to that of taxol in displacement competition assays. Epothilones also cause cell cycle arrest at the G2-M transition leading to cytotoxicity, similar to taxol. In contrast to taxol, epothilones retain a much greater toxicity against P-glycoprotein-expressing multiple drug resistant cells. Epothilones, therefore, represent a novel structural class of compounds, the first to be described since the original discovery of taxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.

L-741,494, a fungal metabolite that is an inhibitor of interleukin-1 beta converting enzyme.

gamma-Pyrone-3-acetic acid (L-741,494) is a novel metabolite produced by a culture of the fungal genus Xylaria. This substance is a water-soluble, competitive, irreversible inhibitor of Interleukin-1 beta Converting Enzyme that is inactive against papain and trypsin. It has a mol wt of 154 and an empirical formula of C7H6O4. We propose the name xylaric acid for this compound.

A light stabilizer (Tinuvin 770) that elutes from polypropylene plastic tubes is a potent L-type Ca(2+)-channel blocker.

A pharmacologically active agent was easily extracted by aqueous or organic solvents from laboratory plastic tubes (Falcon Blue Max) and has been chemically identified as bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate. This compound (approximately 12 micrograms per tube approximately 25 nmol) blocked 1,4-dihydropyridine-sensitive 45Ca2+ uptake into GH3 cells with an IC50 value of 3.6 microM, inhibited Sr2+ currents through L-type Ca2+ channels in A7r5 smooth-muscle cells in whole-cell patch-clamp experiments after extracellular application, and affected the high-affinity binding of Ca2+ entry-blocker ligands to a variety of preparations. Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is a highly potent (IC50 values < 10 nM) inhibitor at the phenylalkylamine- and benzothiazepine-selective drug-binding domains of the alpha 1 subunit of L-type Ca2+ channels. This compound behaves as a heterotropic allosteric regulator for the 1,4-dihydropyridine-selective domain in purified Ca(2+)-channel preparations from rabbit skeletal muscle. (+)-Tetrandrine stimulation of 1,4-dihydropyridine binding to the membrane-bound L-type Ca2+ channel is inhibited by the compound in a competitive manner (Ki value = 6.8 nM). Bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate is therefore classified as the prototype of another class of L-type Ca(2+)-channel blockers that binds to the alpha 1 subunit at the drug-binding domains selective for (+)-tetrandrine or (+)-cis-diltiazem. This compound is identical to Tinuvin 770, which is used worldwide as a light stabilizer for polyolefins.

Pneumocandins from Zalerion arboricola. III. Structure elucidation.

Pneumocandin B0 (6) and six related lipopeptides are antifungal and anti-Pneumocystis carinii agents from mutants of Zalerion arboricola, whose structures were determined mainly on the basis of spectroscopic analysis. They belong, along with pneumocandin A0 (L-671,329) previously isolated from these laboratories, to the echinocandin class of antifungal agents. The product from base-catalyzed ring opening involving the hemiaminal position of the dihydroxyornithine residue of B0, has been clearly defined as 6b. Modifications were limited to the 3-hydroxy-4-methylproline, 3,4-dihydroxyhomotyrosine and 4,5-dihydroxyornithine residues of pneumocandin A0.

Cochinmicins, novel and potent cyclodepsipeptide endothelin antagonists from a Microbispora sp. II. Structure determination.

Cochinmicins I, II, and III are competitive endothelin antagonists produced by Microbispora sp. ATCC 55140. The cochinmicins are cyclic depsipeptides containing six alpha-amino acids and a pyrrolecarboxylic acid. Based upon MS, 1D and 2D NMR, and LC data, the structures and absolute stereochemistries of the cochinmicins have been assigned. All three components have the same basic sequence and contain one equivalent each of D-allo-threonine, D-alanine, L-phenylalanine, D-phenylalanine, 5-chloropyrrole-2-carboxylic acid (or pyrrole-2-carboxylic acid in cochinmicin I), plus two equivalents of 3,5-dihydroxyphenylglycine (DHPG). The phenylalanine residues were differentiated via a methanolysis product which contained only one of the phenylalanine residues. Both DHPG residues have the D configuration in the more active cochinmicins I and III. Cochinmicin II contains both D- and L-DHPG and these residues have been differentiated in the sequence based upon 1H NMR data.

Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated A alpha (1-21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187.

The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood.

Induction of leukocyte activation by meshes surgically implanted in the peritoneal cavity.

The search for surgical prosthetics that do not act to promote infection has been frustrating. However, recent experience with the implantation of polyglycolic acid mesh (PGA) used as an intestinal sling has been associated with a lack of pelvic infections. To examine the basis for these observations, 1 x 1-cm pieces of biomaterials were sewn onto the peritoneal cavity of rats. The biomaterials examined included PGA mesh, a composite mesh composed of a permanent nonabsorbable Novafil inner layer coated with a PGA outer layer, polyvinyl alcohol sponge, and silicon elastomer. Biomaterials were removed at postoperative days 1, 2, 8, and 14, and examined for bacteriostatic and bactericidal activity by standard techniques. Mesh adherent leukocytes were evaluated for their ability to oxidize dichlorofluorescein diacetate (DCFH-DA), which is fluorescent in the presence of intracellular hydrogen peroxide produced by both polymorphonuclear cells and monocytes. PGA and the composite mesh had no intrinsic bacteriostatic or bactericidal activity. However, adherent leukocytes were induced to oxidize DCFH at levels 10-fold and 5-fold, respectively, by postoperative day 14, compared with earlier time points and other biomaterials. The ability of these PGA meshes to stimulate respiratory bursts by peritoneal cells may partly be responsible for the lack of infections associated with their use.

Novel antinematodal and antiparasitic agents from Penicillium charlesii. II. Structure determination of paraherquamides B, C, D, E, F, and G.

Paraherquamides B (2, C27H33N3O4), C (3, C28H33N3O4), D (4, C28H33N3O5), E (5, C28H35N3O4), F (6, C28H35N3O3), and G (7, C28H35N3O4) are novel metabolites of Penicillium charlesii. The structures of these compounds have been determined by NMR and MS analysis.

Differential production of interleukin 1 on the surface of biomaterials.

The production of cytokines on the surface of surgical biomaterials plays a major role in their biocompatibility. Membrane-associated interleukin 1 (mIL-1) is a cytokine found on the surface of macrophages activated by biomaterials. To better understand the host-foreign body interaction, we quantitated the production of mIL-1 on the surface of two materials commonly used in surgery, expanded polytef (ePTFE) and silicon elastomer (SE). The mean (+/- SD) level of mIL-1 produced by adherent cells to ePTFE significantly decreased from day 2 (13,746 +/- 3630 cpm per disk) compared with day 7 (2828 +/- 1304 cpm per disk). However, the level of mIL-1 produced by ePTFE-adherent cells was still markedly greater than the level of mIL-1 produced by cells adherent to SE (1877 +/- 1028 vs 1595 +/- 822 cpm per disk). These results indicate that ePTFE and SE elicit a differential host response in terms of cytokine production. This study may enhance our understanding of the cellular events on the surface of biomaterials that underlie clinical observations.

Cochlioquinone A, a nematocidal agent which competes for specific [3H]ivermectin binding sites.

Cochlioquinone A, isolated from the fungus Helminthosporium sativum, was found to have nematocidal activity. Cochlioquinone A is a competitive inhibitor of specific [3H]ivermectin binding suggesting that cochlioquinone A and ivermectin interact with the same membrane receptor.

Production of hydrogen peroxide by rabbit articular chondrocytes. Enhancement by cytokines.

Recent evidence suggests that reactive oxygen intermediates may play a role in the etiology of cartilage matrix degradation in arthritis. We have previously established that normal articular chondrocytes can functionally act as macrophages. These functions include expression of class II MHC Ag, presentation of Ag and induction of mixed and autologous lymphocyte stimulation. Inasmuch as the production of reactive oxygen intermediates is a hallmark of macrophage activity during inflammatory response, we were interested in examining the ability of normal articular chondrocytes to produce reactive oxygen intermediates. Using the trapped indicator 2',7'-dichlorofluorescin diacetate (DCFH-DA), we measured the levels of intracellular hydrogen peroxide within normal rabbit articular chondrocytes. We found that Concanavalin A induces chondrocytes to rapidly oxidize 2',7'-dichlorofluorescin diacetate to a highly fluorescent dichlorofluorescin in a dose- and time-dependent manner. Fluorescent dichlorofluorescin oxidation by chondrocytes was inhibited by the addition of catalase, an enzyme that detoxifies hydrogen peroxide. Exposure of rabbit chondrocytes to either IFN-gamma or TNF primed the chondrocytes to produce significantly greater amounts of hydrogen peroxide with or without further stimulation. Using scopoletin oxidation as a measure of the release of hydrogen peroxide, we confirmed that chondrocytes released this reactive oxygen intermediate after adherence to serum coated culture plates. Rabbit articular chondrocytes produced and released greater amounts of hydrogen peroxide than pulmonary alveolar macrophages, a well characterized macrophage cell type. These observations suggest that chondrocytes are an important source of reactive oxygen intermediates. Furthermore, the production of reactive oxygen intermediates by chondrocytes may be an important mechanism by which chondrocytes induce structural and functional alterations in cartilage matrix observed during arthritis.