Isolation and Characterisation of an Antifungal Antibiotic (GR135402) with Protein Synthesis Inhibition.

Abstract

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.As part of an antifungal screening programme we searched for compounds from microbial fermentation broths that inhibited fungal protein synthesis as determined by a Candida albicans cell free translation system using polyuridine as a synthetic template. This paper describes the isolation, chemical characterisation and biological properties of the compound designated GR135402 (Fig. 1) Seed cultures were inoculated with two agar plugs added to a 250ml Erlenmeyer flask containing 50ml of medium of the following composition: peptone (Oxoid L34) 10g, malt extract (Oxoid L39) 21g, glycerol 40g, Junlon PW 110 (Honeywell and Stein Ltd, Wallington, Surrey) 1g in 1 litre distilled water. The pH of the medium was adjusted to 6.5 by the addition of aq NaOH before autoclaving. The flasks of inoculated seed medium were at 250rpm with a 50mm throw, for 8 days.The seed cultures were used at 3% (v/v) to inoculate 50ml aliquots of fermentation medium in 250ml Erleneither statically or shaken at 250rpm. Fermenter cultures (5 liters working volume) were aerated at 2.5lpm and stirred at 700rpm. The fermentation medium SM2 contained arkasoy soy flour 15g, glycerol trioleate (Estol 1434) 10g and KH2PO4 1g in 1 litre of tap water. The ingredients were mixed in a Waring blender and the pH adjusted to 7.0 with KOH before autoclaving. Medium SM55 contained maltose 40g, maltodextrin MD30E 120g, peptone (Oxoid L37) 10g, cottonseed flour (Sigma) Fig. 1. Chemical structure of GR135402.

Ribosomal P-protein Stalk Function Is Targeted by Sordarin Antifungals

“…Sordarins are a new family of antifungal compounds and, together with tenuazonic acid (25), the only protein synthesis inhibitors reported to show selectivity between different eukaryotes (17,18). They have been described to target and bind to elongation factor 2 1,2 (18), but it was also immediately apparent that the inhibitor's mode of action involved additional cellular components, because resistant mutants were obtained outside EF2 1 , and high affinity binding of the drug to macromolecules required the presence of ribosomes, 2 suggesting that the binding site on EF2 is fully formed only after the factor interacts with a ribosome.…”

“…Semisynthetic derivatives of the natural product sordarin (15,16) have proven to be highly potent inhibitors of eukaryotic protein synthesis, with remarkable selectivity for the fungal translation machinery (17,18). Sordarin inhibitors seem to bind to elongation factor 2 1,2 (18), and point mutations on the factor make cells resistant to the inhibitors 1 (18), but high affinity binding requires the presence of ribosomes, 2 and resistant mutants were detected, which did not map on EF2.…”

A Chemical Genomic Screen in Saccharomyces cerevisiae Reveals a Role for Diphthamidation of Translation Elongation Factor 2 in Inhibition of Protein Synthesis by Sordarin

“…A family of sordarin-related compounds has been described, all of them inhibiting cell growth by interfering with protein synthesis (6,7). Even though there is a high degree of homology between the fungal and mammalian protein synthesis machineries, these inhibitors are highly specific for the fungal elongation step (24). Moreover, the sordarin derivatives display various levels of antifungal activity when tested against different species of fungi, with some of them being sensitive while others are resistant (15,28).…”

Applications of yeast in drug discovery

“…This class of compounds was first described as antifungal agents in 1970 [85,86] and was rediscovered in a screen designed to isolate fungal-specific protein synthesis inhibitors [87]. Previous to Kinsman et al [87], the mode of action of this class of antifungals had not been examined.…”

“…This class of compounds was first described as antifungal agents in 1970 [85,86] and was rediscovered in a screen designed to isolate fungal-specific protein synthesis inhibitors [87]. Previous to Kinsman et al [87], the mode of action of this class of antifungals had not been examined. No derivatives of sordarins are on the market yet but some sordarin derivatives, such as GM 222712 and GM 237354, show excellent in vitro activities against a diverse set of pathogenic fungi, including Candida spp., C. neoformans, Pneumocystis carinii and some filamentous fungi [88].…”