Increased antifungal activity of L-733,560, a water-soluble, semisynthetic pneumocandin, is due to enhanced inhibition of cell wall synthesis

Abstract

The pneumocandins are natural lipopeptide products of the echinocandin class which inhibit the synthesis of 1,3-p-D-glucan in susceptible fungi. The lack of a corresponding pathway in mammalian hosts makes this mode of action an attractive one for treating systemic infections. Substitution by an aminoethyl ether at the hemiaminal and dehydration and reduction of the glutamine of pneumocandin Bo produced a semisynthetic compound (L-733,560) with intrinsic water solubility, significantly increased potency, and a broader antifungal spectrum. To evaluate the mechanism for the improved antifungal efficacy, we determined that L-733,560 was a more potent inhibitor of glucan synthase activity in vitro, did not affect the other membrane-bound enzymes tested, conferred susceptibility to lysis in the absence of osmotic support, and did not disrupt currents in liposomal bilayers or 86Rb+ fluxes from liposomes. In AspergiUus species L-733,560 also produced the same morphological alterations as pneumocandin Bo. A stereoisomer of L-733,560 with poor antifungal activity was a weak inhibitor of glucan synthase. All of these results support the notion that the enhanced antifungal activity of L-733,560 is achieved by superior inhibition of glucan synthesis and not by nonspecific membrane effects or a second mode of action.The echinocandins, pneumocandins, and papulacandins are antifungal agents which inhibit the synthesis of 1,3-,3-D-glucan in susceptible organisms (3,26,27,35,(38)(39)(40)42). Because the likelihood of mechanism-based toxicity is reduced, inhibition of the synthesis of a fungus-specific structure is an attractive mode of action for antifungal drug candidates. In addition to inhibition of in vitro glucan synthase activity, several observations on whole cells support the notion that these compounds act to inhibit cell wall synthesis in intact fungi. First, osmotic support prevents the lysis of cells treated with drug at the MICs (13, 45). Since the intact fungal cell wall counteracts the high turgor pressure of the protoplast, an agent which disrupts cell wall integrity produces membrane swelling and 'ultimately causes lysis in the absence of osmotic support. Second, susceptible organisms undergo gross morphological changes after treatment with these agents, consistent with cell wall alterations (10,13,16,22,28,33,44). Third, whole-cell labeling experiments which monitor macromolecular synthesis demonstrate the preferential inhibition of cell wall synthesis (3, 13). Lastly, Douglas et al. (21) recently showed that a mutation responsible for pneumocandin resistance in Saccharomyces cerevisiae cosegregates with enzyme activity resistant to inhibition by the pneumocandins.Originally, semisynthetic derivatives of the natural products were screened for reduced erythrocyte lysis, and this led to the development of the echinocandin B-based analog cilofungin (26,27 cantly alter the in vitro potency of the lipopeptide against Candida species compared with that of the natural product (17). Newer side chain analogs of the echi...

Cloning and characterization of GNS1: a Saccharomyces cerevisiae gene involved in synthesis of 1,3-beta-glucan in vitro

“…Despite the fungicidal activity of L-733,560, the S. cerevisiae ␤-glucan synthase is much more resistant to the drug by comparison with the Candida albicans or Aspergillus fumigatus enzymes (27). The drug also achieves a 50% inhibition of the S. cerevisiae enzyme at relatively higher concentrations than its MIC against whole cells (14,16,27).…”

Differential Expression and Function of Two Homologous Subunits of Yeast 1,3-β-D-Glucan Synthase

“…We have previously shown that FKS1 is likely to encode a subunit of 1,3-␤-D-glucan synthase (18). This conclusion was based primarily on the observation that certain mutations in FKS1 result in whole-cell resistance to the glucan synthase inhibitor L-733,560, a member of the echinocandin class of antifungal agents (45,72), as well as in glucan synthase activity which is resistant to L-733,560 in vitro (19,20). The high degree of similarity between Fks2p and Fks1p indicates that Fks2p is also likely to be a subunit of this enzyme.…”

“…provides further evidence that the antifungal antibiotics such as the pneumocandins, echinocandins, and papulacandins, which are inhibitors of glucan synthase, can act solely by inhibiting this enzyme (45,72). Moreover, it appears that in S. cerevisiae both forms of the enzyme must be inhibited by L-733,560 to achieve a fungicidal effect.…”

Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties

“…The final concentration of test compounds ranging from 64 g/ml to 0.06 g/ml in 100 l of sterile saline containing 3.2% Me 2 SO was prepared in 96-well Microtest U-bottom plates (BD-353227; Becton Dickinson). The RBC lysis assay (32) was initiated by addition of 5 l of 3% washed human RBC suspension to the each well in the plates. The plates were then immediately shaken for 1-2 min at 550 rpm to disperse the RBCs and incubated at room temperature for 60 min.…”