L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. I. The producing organism and its fermentation.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.

A structurally unique, potent, and selective oxytocin antagonist derived from Streptomyces silvensis.

The in vitro and in vivo oxytocin/arginine vasopressin (OT/AVP) antagonist properties of two cyclic hexapeptides derived from a newly discovered natural product (L-156,373) of Streptomyces silvensis are described. In radioligand binding assays, L-156,373 [cyclo(L-Pro-D-Phe-N-OH-L-Ile-D-piperazyl-L-piperazyl-N-Me-D -Phe)] exhibited moderate affinity for rat uterine OT receptors (Ki, 150 nM), with some selectivity (approximately 20-fold) vs. liver AVP-V1 and kidney AVP-V2 receptors. Dehydroxylation of N-hydroxyisoleucine and oxidation of the piperazic acid residues of L-156-373 produced an interesting derivative, L-365,209. These structural modifications increased OT receptor affinity and selectivity by 20- and 2.5-5-fold, respectively. In the isolated rat uterus, L-365,209 was a potent (apparent dissociation constant, 1.7 nM) and competitive OT antagonist. L-365,209 also blocked the effects of AVP at both AVP-V1 (phosphatidylinositol turnover in rat hepatocytes) and AVP-V2 (adenylate cyclase in rat kidney medulla) receptors, but only at low micromolar concentrations. L-365,209, given iv to anesthetized rats, antagonized the action of exogenous OT on the uterus (ID50, 460 micrograms/kg) with a relatively long duration of action. L-365,209 represents a unique class of compounds that provides an entirely new approach for the design of antagonists for these neurohypophyseal hormones.

Quaternary heterocyclylamino beta-lactams. III. The mode of action of L-640,876 and the effect of NaCl on membrane permeability and binding.

L-640,876, 7-beta(1-benzylpyridinium-4-yl)amino-3-[( (1-methyl-1 H-tetrazol-5-yl)thio]methyl)-ceph-3-em-4-carboxylate, is a potent representative of a new family of beta-lactam antibiotics which are similar in some respects to mecillinam. When L-640,876 and mecillinam were compared for effects on growth and morphology of Escherichia coli, it was observed that both drugs caused the formation of lemon-shaped cells during the first 30 minutes of exposure and during this period the culture turbidity increased without an appreciable change in culture viability. Unlike mecillinam, after 60 minutes of exposure to L-640,876 the majority of the lemon-shaped cells transformed into spindle-shaped cells and in the continuing presence of the drug formed osmotically fragile spheroplasts. Membrane binding studies indicated that, like mecillinam, L-640,876 was bound to the PBP-2 of E. coli and Proteus morganii; however, some binding of L-640,876 to the PBP-3 of E. coli was detected. In Staphylococcus aureus binding differences were more evident as L-640,876 was more rapidly bound to PBP-1 and 2 whereas mecillinam was rapidly bound to PBP-3. The reversal of inhibition of certain strains of Gram-negative bacteria by high ionic strength media could not be directly attributed to a reversal of antibiotic binding to the PBPs. Permeability studies indicated that the superior potency of L-640,876 in E. coli was partly due to its higher concentration in the periplasm which was unaffected by the simultaneous addition of drug and NaCl, however, in cells cultured in high ionic strength medium there was a marked reduction in penetration rate of all beta-lactams tested.

Quaternary heterocyclylamino beta-lactams. II. The in vitro antibacterial properties of L-640,876, a new type of beta-lactam antibiotic.

A new semisynthetic cephalosporin antibiotic designated 7-beta-(1-benzylpyridinium-4-yl)-amino-3-[( (1-methyl-1H-tetrazol-5-yl) thio]methyl)ceph-3-em-4-carboxylate (L-640,876) was compared for antibacterial activity in vitro with mecillinam, cefoxitin and cefotaxime. The antibacterial spectrum of L-640,876 and the effect of culture medium composition and inoculum size on activity are most similar to those of mecillinam. In some cases the inoculum effect on MICs correlated with instability of the compound to certain beta-lactamases and in others to the presence of ionized compounds such as sodium chloride in the medium. On balance, L-640,876 was superior to mecillinam in potency and breadth of spectrum.

Production and partial purification of Salmonella enterotoxin.

By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin.

Assay, characterization, and localization of an enterotoxin produced by Salmonella.

An enterotoxic factor isolated from cultures of Salmonella yielded reproducible results in the suckling mouse model in contrast to other animal models. The enterotoxin appears to possess properties similar to both the heat-stable and heat-labile enterotoxins of Escherichia coli. Preliminary results indicate that the toxin is a protein, is located in the cell wall or outer-membrane fraction, and is difficult to separate from other cell wall constituents.