A novel class of antibiotics was isolated from cultures of Streptomyces coeruleorubidus strain AB1183F-64. The antimicrobial activity of the pacidamycins is selective against Pseudomonasaeruginosa. The various congeners are nucleoside peptides which differ in the terminal amino acid residues. The structures were determined using MS-MSand 2D NMR techniques.

Pacidamycins are nucleosidyl-peptide antibiotics which have activity only against Pseudomonas aeruginosa. Their MICs for other organisms such as Enterobacteriaceae, Staphylococcus aureus, most Streptococci and other Pseudomonas species are > 100 iwg/ml. These compounds had no activity against erythromycin-susceptible Streptococci. The MICs forStreptococcus pyogenes with constitutiveand inducible-type of macrolide-lincosamidestreptogramin resistance were 12.5 and 25 jwg/ml, respectively. The MICs against P.aeruginosa ranged from 8 to 64^g/ml. The activity of these compounds was 1 to 2-fold less in serum than broth. Time-kill curves were performed using 4 and 8 times the MIC of pacidamycin 1. It was bactericidal against P. aeruginosa (3 log10 decrease in 4 to 6 hours). At 24 hours, resistant mutants were found in the cultures. The MICsof piperacillin and gentamicin for these mutants were the same as for the parent strain. The frequency of resistance to these compounds was<3.5x lO~6. The resistant mutants were stable after 10 transfers in antibiotic-free medium.The pacidamycins were inactive against P. aeruginosa in mouseprotection tests. After a single subcutaneous injection of25 mg/kg ofpacidamycin 1, the Cmaxwas approximately 50 jugjml and the serum half-life was 0.5 hour.Pseudomonas aeruginosa infections are a problem to treat in spite of the availability of large numbers of antibiotics because of their intrinsic resistance and their ability to become resistant to new classes of antibacterial agents1}. Pacidamycins are a novel class of antibiotics which have been isolated from a screen designed to find antibiotics with activity against P. aeruginosa. The taxonomy of the producing culture, the fermentation, isolation and chemical structures of these compounds are described in the preceding manuscripts2>3). The biological activities of these compounds will be described in part at the 28th Intersci. Conf. on Antimicrob. Agents Chemother.4). Materials and Methods AntibioticsThe pacidamycins were isolated at Abbott Laboratories. The majority of the experiments were performed with pacidamycin 1 (A-68567), the alanine-tryptophan analog. Pacidamycins 2 (alaninephenylalanine analog), 3 (alanine-meta-tyrosine) and 5 (phenylalanine analog) were also tested when sufficient quantities were availableIn Vitro Antibacterial Activity The bacterial strains used in this study were clinical isolates or cultures obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, U.S.A.) which are maintained frozen in our laboratory. MICswere determined by the agar dilution method using brain heart infusion agar and Mueller-Hinton agar5).

The novel antifungal agents, calbistrins A, B, C and D have been isolated from a strain of Penicillium restrictum (AB 1875C-28). The four congeners were separated by bioactivity directed fractionation using countercurrent chromatography and preparative-HPLC. NMRstudies revealed that the calbistrins each contain a carboxylic acid conjugated tetraene attached through an aliphatic ester linkage to a hexahydronaphthalene system. 39In the course of screening microorganisms for the production of bioactive metabolites, a penicillium was discovered which produced potent activity against Candida albicans. Bioactivity directed fractionation of a fermentation extract provided four new related antifungal agents. The majority of the bioactivity was found in the mycelia, which was filtered off and extracted with acetone. The concentrated extract was partitioned between ethyl acetate and water, CHC13-MeOH-H2Oand methanol-w-heptane. The four calbistrins were recovered as a mixture from the methanol solubles by droplet countercurrent chromatography. They were then separated and further purified by preparative-HPLC using a silica-bonded phenyl column.High resolution FABmass spectrometry in the positive ion mode gave an (M+Na)+ parent mass of 563.2624 for calbistrins A and B, and 565.2774 for C and D; indicating molecular formulae for A and B of C31H40O8and for C and D of C31H42O8.NMRstudies revealed that the calbistrins each contain a carboxylic acid conjugated tetraene which differentiates the isomers A from B, and C form D. In each compoundthis conjugated olefin is attached through an aliphatic ester linkage to a hexahydronaphthalene system. In calbistrins A and B, the hexahydronaphthalene system is fused with a cyclic hemiacetal. In calbistrins C and D this group has been reduced to the open ring diol. Calbistrins A and B exhibit potent activity against Candida albicans and somerelated species. Calbistrins C and Dare less active. The structural characterization of the calbistrins is outlined in this paper. Microbiological and fermentation data are presented in a comparisonpaper.l) Isolation The isolation of the calbistrins was guided by the use of disc diffusion bioassays on agar plates seeded with Candida albicans. Ninety liters of whole beer was filtered through glass wool impregnated paper and the filter cake was dispersed and soaked in sixteen liters of acetone for 16hours (at 4°C). After additional stirring, the mixture was filtered through glass wool impregnated paper and the filter cake was washed with an additional eight liters of acetone. The acetone extract was concentrated on a circulating flash evaporator until approximately four liters of residual aqueous acetone remained. This was extracted with

Twonovel triterpene sulfates have been isolated from Fusarium compactumby bioactivitydirected fractionation using an assay which measures the inhibition ofproteolytic activity ofrhinovirus 3C protease on a fluorogenic peptide substrate. The compounds were purified by countercurrent and reverse phase chromatographies. NMR,MS, UVand IR studies revealed two triterpene sulfates, uncommon metabolites of terrestrial fungi.In the course of screening microorganisms for the production of bioactive metabolites, a Fusarium compactum sp., isolated from an ant hill soil sample collected near the village of Rantan, Nigeria, was found to produce rhinovirus 3C protease inhibition activity. The 3C protease, one of two virally encoded proteases, is responsible for cleaving the initially translated rhinoviral polyprotein at eight locations to form the mature structural proteins and viral replicative enzymes. 1} There are more than 100 known serotypes of human rhinovirus,2) and they are thought to be responsible for 40~60% of the common colds.3) In principle, compoundswhich inhibit the proteolytic action of the 3C protease would disrupt replication of the rhinovirus, and could lead to an effective drug treatment for the common cold.Using an assay which measures the inhibition of 3Cprotease activity on a synthetic fluorogenic substrate, bioactivity-directed fractionation of a stationary fermentation extract of Fusarium compactum (AB 21941-103) provided two triterpene sulfates. Triterpene sulfates had not been reported as bioactive metabolites from terrestrial fungi until Vesonder and Burmeister isolated them from several Fusarium species in a search for potentially phytotoxic secondary metabolites.4'5) It was found that these metabolites are produced in relative abundancewhenterrestrial Fusarium species are grown on grain substrates.5) In our study, Shredded Wheat was used as a solid support for the growth of the fungus.

The novel calcineurin inhibitor, dibefurin, has been isolated from the fungal culture AB 16501-759. The isolation was bioactivity-directed fractionation using an assay which measures the phosphatase activity of calcineurin. The compoundwas purified by countercurrent, reverse phase and gel filtration chromatographies. Several studies, including crystallographic, NMRand MS, revealed that dibefurin is a novel dimeric compoundof a unique structural type.In the course of screening microorganisms for the production of bioactive metabolites, a fungal culture (AB 16501-759) was discovered which was found to produce calcineurin inhibition activity. Calcineurin phosphatase is a calcium/calmodulin -regulated serine/ threonine phosphatase, which plays a key role in T cell activation. It has been shown that calcineurin dephosphorylates the cytosolic component of the NFAT complex, and thereby regulates nuclear translocation and dimerization.1'2) This process is necessary for the production of interleukin-2 and other T-lymphocyte derived cytokines. The commontarget of the immunophilin complexes of both cyclosporin A and tacrolimus is thought to be calcineurin.3) Compoundsfound in a screen for direct calcineurin inhibitors would be expected to have immunosuppressant activity independent of the requirement for cyclophilin or FKBP. Direct calcineurin inhibitors may be useful in distinguishing the immunosuppressive activity of such agents from any complications which may arise from inhibition of the peptidyl prolyl isomerase activity of the immunophilins.Using an assay for the phosphatase activity of calcineurin, bioactivity-directed fractionation of an extract of a stationary fermentation of AB 16501-759 provided a novel microbial metabolite of a unique dimeric structural type (1), dibefurin. Dibefurin appears to be a dimerization derivative of 4,5,6-trihydroxy-7-methyl-(l//,37/)-dihydroisobenzofuran.Both the monomer and the dimer are novel. The monomerfrom which dibefurin is derived, could itself be a derivative of flavipin (2) Phenolic substances are commonlyencountered as mold metabolites.5) The structure of dibefurin was elucidated mainly by NMR,MSand crystallographic studies. The dibefurin producing strain characterization, fermentation, isolation, structural determination and some biological data are outlined in this paper. Characterization of the Producing StrainThe appearance of colonies of strain AB16501-759 was quite different whenthe culture was grownunder continuous fluorescent light than when grown in the dark. Although the colonies were usually quite irregular in the dark, the culture formed regular, round colonies when incubated in the light. Colonies grew faster in the light. Also, the mycelium had a sectored appearance in the dark, and different pigments were produced under each incubation condition. Strain AB16501-759 formed a soluble brown pigment on potato dextrose agar (PDA, Difco) in the dark but not in the light. No reproductive structures were ever observed under any growth con- was 42~45 mm.Colonies on ...