Evaluation of cyclooxygenase-2 inhibitors using pulsed ultrafiltration mass spectrometry.

Since selective inhibition of the inducible form of cyclooxygenase (COX-2) might retain all the benefits of classical nonsteroidal antiinflammatory agents while avoiding the major side effects associated with inhibition of the constitutive isoform COX-1, COX-2 has become an important target for the discovery and development of new antiinflammatory drugs. To aid in the discovery and characterization of such selective inhibitors, we have applied a mass spectrometry-based screening technique, pulsed ultrafiltration mass spectrometry, using COX-2 as the target. In a blind study, 18 samples enriched with one or more inhibitors of COX-2 were evaluated. The matrixes for the test samples consisted of DMSO, r DMSO solutions of a plant extract, or a bacterial fermentation broth extract. The composition of the samples was unknown during the assays, as were the concentrations of the COX-2 inhibitors. A soluble recombinant form of human COX-2 was incubated with each sample, and then an aliquot of each mixture was injected into the stirred ultrafiltration chamber fitted with a 30000 MW cutoff ultrafiltration membrane. After the unbound and weakly bound compounds were washed away, the ligand-receptor complexes were disrupted using an acidified 10% methanol solution. The released ligands were trapped on a C18 cartridge and then identified using liquid chromatography-negative ion electrospray mass spectrometry with the trapping cartridge as the HPLC column. Neither the plant matrix nor the fermentation broth extract were found to interfere with the assay. Two or three ligands for COX-2 were identified in each sample, which included polar and nonpolar compounds and inhibitors with IC50 values ranging from 100 microM to 10 nM.

Structure-activity relationship studies on 1-[2-(4-Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.

Leukotriene B(4) (LTB(4)) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA(4) hydrolase is the rate-limiting step for LTB(4) production, this enzyme represents an attractive pharmacological target for the suppression of LTB(4) production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA(4) hydrolase. Structure-activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA(4) hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

O- and N-methylation in the biosynthesis of staurosporine.

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.

Four new clerodane diterpenes from the leaves of Casearia guianensis which inhibit the interaction of leukocyte function antigen 1 with intercellular adhesion molecule 1.

Four new clerodane diterpenes, casearinols A and B (1 and 2) and casearinones A and B (3 and 4), were isolated from the leaves of Casearia guianensis. These immunomodulatory compounds have been structurally elucidated primarily on the basis of 2D NMR analysis and spectral data comparison with known compounds. These compounds inhibited the binding of T-cell leukocyte function antigen 1 to intercellular adhesion molecule 1.

Isolation and structure of leukotriene-A4 hydrolase inhibitor: 8(S)-amino-2(R)-methyl-7-oxononanoic acid produced by Streptomyces diastaticus.

The novel amino acid 8(S)-amino-2(R)-methyl-7-oxononanoic acid (1) was isolated from the soil-borne microorganism Streptomyces diastaticus during our screening for inhibitors of leukotriene-A4 hydrolase (LTA4H), a requisite enzyme in the biosynthesis of the potent inflammatory mediator leukotriene-B4 (LTB4). The structure of 1 was determined by detailed spectroscopic analyses and is related to 7-keto-8-aminopelargonic acid (2), a biosynthetic precursor of biotin. The relative potency of 1 (LTA4H IC50 = 0.6 microM) warranted further biological studies.

Isolation and structure of harzianum A: a new trichothecene from Trichoderma harzianum.

A new trichothecene, harzianum A [1], was isolated from the soil-borne fungus Trichoderma harzianum. The structure of 1 was determined by extensive spectral analyses including the nmr techniques of PS-COSY, HMQC, HMBC, and NOESY. Harzianum A [1] contains a (Z,E,E)-2,4,6-octatriendioic acid esterified on the 4 beta hydroxyl group of trichodermol and is structurally related to the trichoverroids. Harzianum A [1] showed no cytotoxicity against baby hamster kidney cells, no activity against Gram-negative and Gram-positive bacteria, but modest antifungal activity at 100 micrograms/ml.

Cytotoxicity of trichothecene mycotoxins isolated from Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum in baby hamster kidney (BHK-21) cells.

Twenty-six trichothecene mycotoxins produced by Fusarium sporotrichioides (MC-72083) and Fusarium sambucinum were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-21) cells. The relative cytotoxicity was measured as LC100. The most cytotoxic trichothecenes were T-2 toxin (5 ng/ml) and the recently isolated 4-propanoyl HT-2 (5 ng/ml) and 3'-hydroxy T-2 toxin (5 ng/ml). T-2 tetraol (1 x 10(4) ng/ml), 8-beta-hydroxytrichothecene (1 x 10(4) ng/ml), sporotrichiol (2 x 10(4) ng/ml), 8-oxodiacetoxyscirpenol (6 x 10(4) ng/ml) and 8-acetyl T-2 tetraol (1 x 10(5) ng/ml) were the least toxic of the regular trichothecenes. None of the modified trichothecenes or the apotrichothecene were very cytotoxic: 8-beta-hydroxysambucoin (2 x 10(3) ng/ml), FS-1 (5 x 10(3) ng/ml), 8-alpha-hydroxysambucoin (8 x 10(4) ng/ml) and trichotriol (1 x 10(5) ng/ml). The modified trichothecenes, FS-2 and FS-3, were not toxic even at 1 x 10(5) ng/ml. The baby hamster kidney cell bioassay proved to be a very sensitive and reproducible means of screening new trichothecene mycotoxins for relative cytotoxicity.

Absolute and relative configuration of erythroskyrin.

We have reisolated erythroskyrin from Penicillium islandicum and have determined the relative stereochemistry of the compound through extensive 1H- and 13C-nmr studies. The absolute stereochemistry was determined by nmr studies of the O-methylmandelate esters.

Isolation and characterization of 4'-hydroxy T-2 toxin, a new metabolite of the trichothecene mycotoxin T-2.

Biotransformation of the trichothecene mycotoxin T-2 by the hepatic S-9 fraction prepared from phenobarbital-treated rats yielded a new metabolic product designated RLM-3. The metabolite was purified from the hepatic preparation using preparative HPLC. The structural analysis of RLM-3 was carried out using gas chromatography/mass spectrometry and 1H and 13C NMR. RLM-3 was identified as 4'-hydroxy T-2. The toxicity of RLM-3 in comparison to T-2 toxin and 3'-hydroxy T-2 was assessed using a rat skin bioassay technique. The metabolite 4'-hydroxy T-2 was shown to be deacylated at the C-4 position to yield 4'-hydroxy HT-2 when incubated with rat hepatic S-9 preparations.

Secondary metabolites from Fusarium. Two new modified trichothecenes from Fusarium sporotrichioides MC-72083.

Two new, relatively non-toxic, secondary metabolites characterized as 8 alpha- and 8 beta-hydroxysambucoin [1] and [2], isolated from the toxigenic fungus Fusarium sporotrichioides MC-72083 are reported. The structural assignments were established by spectral data with 1H-nmr studies (COSY, dnOes) playing a key role in establishing the stereochemistry in 1 and 2. The isolation of 14 known trichothecenes produced by this fungus is also discussed.

Mutagenic perylenequinone metabolites of Alternaria alternata: altertoxins I, II, and III.

The mold genus Alternaria is a widely distributed plant pathogen. Some of these species, e.g., A. alternata, are common decay organisms of fruits and vegetables. Two novel perylene oxide metabolites, altertoxins II and III, have been identified in extracts of A. alternata isolates that exhibit mutagenic responses in the Ames Salmonella typhimurium assay. These identifications were based on mass, optical rotational, and 1H- and 13C-nmr spectral studies. Previous reports of related perylene dione mycotoxins have been clarified.