The gene encoding M 2 , the ion channel-forming protein of influenza virus A, was expressed under the control of an inducible promoter in Saccharomyces cerevisiae. By using single and multicopy plasmids containing GAL promoter-M 2 fusions, a correlation was observed between plasmid copy number and growth in medium inducing M 2 expression. Cells expressing M 2 from multicopy plasmids have reduced growth rates, suggesting that high levels of M 2 are toxic to growth. The addition of amantadine, a compound known to block the ion channel activity of certain M 2 alleles, restores the growth rates to wild-type levels in cells expressing an amantadine-susceptible allele of M 2 but not an amantadine-resistant allele of M 2 , suggesting that M 2 expression in S. cerevisiae results in the formation of functional M 2 ion channels. Measurements of extracellular acidification by microphysiometry suggest that proton efflux in M 2 -expressing cells is altered and that the addition of amantadine permits the reestablishment of the proton gradient. The growth impairment phenotype resulting from M 2 expression was used to develop a high-capacity screening assay which identified a novel inhibitor possessing an antiviral profile similar to that of amantadine.Functional expression of ion channels in heterologous cell types provides a general method for studying the properties of channel function. Expression in Xenopus laevis oocytes has provided numerous insights into ion channel function (17,18,35). More recently, expression of ion channels in Saccharomyces cerevisiae strains defective in K ϩ uptake has served as a means for isolating new genes encoding functional channels (1, 29). However, expression of ion channels in yeast cells is not restricted to the restoration of uptake functions and may result in a variety of phenotypes, depending on the properties of a particular channel.The M 2 protein of influenza virus A is one of three integral membrane proteins contained in the viral lipid envelope. The M 2 protein is a 97-amino-acid polypeptide containing a single membrane-spanning region. M 2 polypeptides associate as disulfide-linked homotetramers to form ion channels (15,34). Direct evidence defining the ion channel function of M 2 has recently been obtained by expression studies in Xenopus oocytes (26, 38) and in in vitro studies (6,28,36). A therapeutic agent for influenza virus A infections, amantadine, has been shown to function by blocking M 2 ion channel activity (7,12,13,26,32,33,37,38). Other biophysical studies have confirmed that the transmembrane portion of the molecule is the binding site and suggest that the mechanism of action is binding of the compound within the channel pore (6,7,27). Inhibition studies with amantadine suggest that M 2 is required at both early and late stages in the infection cycle of the virus. Early in infection, M 2 permits the flow of protons from the endosome into the virion. The resultant decrease in pH facilitates the dissociation of the matrix protein (M 1 ) from viral genomic ribonucleopro...

Based on the structure-activity relationship data of BMY-28864 and related pradimicin derivatives, the calcium salt-forming ability and the D-mannopyranoside-speciflc visible absorption maximumshift of BMY-28864 were analysed in the ternary complex formation of BMY-28864 with D-mannopyranoside and calcium. The free C-18 carboxyl group of BMY-28864 was proved to be the sole site for binding to calcium, while no hydroxyl groups of the aglycone were involved in calcium salt formation. The stereospecific D-mannopyranoside-recognizing ability of BMY-28864 was completely abolished by removal of the C-5 disaccharide moiety, and, more particularly, of the C-5 thomosaminemoiety. Close relationship of these findings with the antifungal action was also supported by the in vitro antifungal assay and the potassium leakage induction test.In previous papers1~4), the in vitro antifungal activities of pradimicin and benanomicin derivatives on yeasts were shown to be specifically expressed only in the presence of calcium. Using BMY-28864, a water-soluble pradimicin derivative, specific binding of the pradimicin to yeast cells was proved to depend on the ternary complex formation of BMY-28864with mannan and calcium at a molar ratio of 2 :4 : 15). This highly stereospecific binding of BMY-28864 to the mannose unit (more generally, the specific sugar-recognizing ability of the pradimicin and benanomicin family of antibiotics) is biochemically worth studying, as it is currently unexplicable by the widely accepted concepts of receptor-ligand binding in the light of lectin and carbohydrate sciences. Lectins have been considered to recognize specific sugars based on the intrinsic properties of their peptide components, whereas the pradimicin and benanomicin family of compounds are not peptides. Under these circumstances, it is crucially important and essential to more precisely elucidate the mechanismof ternary complexformation of pradimicins with specific sugars and calcium in critical comparison with lectins. This type of knowledge is not only biochemically useful for receptology, but also clinically important from the viewpoint of selective toxicity of final pradimicin drugs in hosts, as sugars are essential cellular components of host animals to be treated with pradimicin, and assumed to exist ubiquitously at significant concentrations in a variety of forms throughout therapy.In this paper, the structure-activity relationship of BMY-28864 and related pradimicin derivatives is analyzed for identification of the moieties of BMY-28864responsible for binding to D-mannopyranoside and calcium and for induction of the visible absorption maximumshift. In brief, only the free C-18 carboxyl group of BMY-28864serves to bind to calcium as salt, while the C-5 disaccharide moiety is essential for specific recognition of and binding to D-mannopyranoside.

The structures of new antitumor antibiotics, glidobactins A (la), B (Ib) and C (Ic) were elucidated by a combination of chemical and enzymatic degradations and spectral analyses. They have in common a cyclized tripeptide nucleus composedof L-threonine, 4(S)-amino-2CE>pentenoic acid and (?ry^r

Cispentacin (( -)-(li?,2^)-2-aminocyclopentanel -carboxylic acid) is a new antifungal antibiotic possessing potent anii-Candida activity. The 50% inhibitory concentration (IC50) and IC100 values of cispentacin against clinical isolates of Candida albicans were in the ranges 6.3~12.5 and 6.3~50/jg/ml, respectively, by turbidimetric measurement in yeast nitrogen base glucose medium. No significant activity was seen against any yeasts and molds whentested by the agar dilution method using three different agar media; Knopp's agar, yeast extract -glucose -peptone agar and Sabouraud dextrose agar. This antibiotic demonstrated good therapeutic efficacy against a systemic Candida infection in mice by both parenteral and po administrations. The 50%protection dose (PD50) values after single iv and po administrations were 10 and 30 mg/kg, respectively. It was also effective in a systemic infection with Cryptococcus neoformans and in both lung and vaginal infections with C. albicans in mice. Cispentacin did not induce acute lethal toxicity at 1,000mg/kg by iv injection and 1,500mg/kg by ip and po administrations in mice.

A variety of glidobactin analogs modified at the fatty acid, L-threonine and nucleus moieties of the moleculeweresynthesized and their structure-activity relationships examined. The antitumor and antifungal activity was greatly influenced by modification of the fatty acid glidobactin, with the dodecanoyl and tetradecanoyl analogs exhibiting better antitumor activity than the parent antibiotics. Replacement of the L-threonine with other amino acids greatly reduced the activity and reduction of the double bond of the nucleus completely eliminated the biological activity of glidobactin. Glidobactin is a complex of new antitumor antibiotics produced by Polyangium brachysporum No. K481-B101 (ATCC 53O8O)1). The three components, glidobactins A, B and C were isolated and their unique acylpeptide structures were elucidated by a combination of chemical and enzymatic degradation and spectral analysis2). The glidobactins inhibit growth of fungi and tumor cells and markedly prolong the life span of mice implanted with P388 leukemia. The strong antitumor activity and unusual structures of glidobactin prompted us to modify the antibiotics. Upon treatment with papain or ficin, glidobactin A (la) was cleaved to give the acyl-L-threonine (2) and the cyclic amine (glidobamine, 3) (Scheme 1). On the other hand, the acylase of Pseudomonas sp. hydrolyzed la to yield (E,E)-2,4-dodecadienoic acid (4) and L-threonylcyclic amine (glidobactamine, 5)3). These hydrolysis products provided appropriate starting materials for modification of the antibiotic. Wereport here the preparation, physico-chemical properties and biological activity of 32 glidobactin derivatives. Chemistry Modification of the fatty acid moiety of glidobactin was carried out by two routes (Scheme 2). Acylation of 5 with appropriate active esters, anhydrides or halides afforded glidobactin acyl analogs in good yields (Scheme 2, route a). Alternatively, coupling of appropriate acid with L-threonine by active ester method yielded acyl-L-threonines which were condensed with 3 to afford the fatty acid derivatives of glidobactin (route b). Replacement of the L-threonine with other amino acids was carried out by route b. The appropriate amino acids were acylated with 4 and the resulting (i^F^^-dodecadienoylamino acids were then coupled with 3 to give the amino acid derivatives. For L-lysine and L-glutamic acid deriva