Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins

Abstract

Polyoxin D, nikkomycin X, and nikkomycin Z are all competitive inhibitors of chitin synthetase 2 (Chs2), the essential enzyme for primary septum formation in Saccharomyces cerevisiae, and of Chs1, a repair enzyme. However, Chs2 is more resistant to these antibiotics than Chs1. When Co2+, the best stimulator of Chs2, was used in the assay for this enzyme, the differences in the Ki values for nikkomycins between the two isozymes reached 3 orders of magnitude. These results point to differences in the active sites of the two isozymes. Polyoxin D was much more effective than nikkomycin Z in inhibiting cell growth. This underlines the importance of the choice of enzyme and of assay conditions when cell wall-synthesizing enzymes are used in screens for possible antifungal agents.

Isolation of canCHS1A, a variant gene of Candida albicans chitin synthase

“…The K, value for UDP-GlcNAc was 0.4 mM for canChslA with Mg2+ (data not shown), in good agreement with the values reported for other chitin synthases; 0.5 mM for sacChsl with Mg2+, 0.65 mM for sacChs2 with Co2+, and 0.28 mM for sacChs2 with Mg2+ (Cabib, 1991).…”

“…Apparent Ki values of polyoxin D and nikkomycin 2 for canChsl A were approximately 5.2 pM and 12 pM, respectively. Dixon plot analysis indicated that both compounds inhibited competitively and that their calculated Ki values were closer to those of sacChs2 than to those of sacChsl (Cabib, 1991). when Mg2+ was present.…”

Cloning of a Candida albicans peptide transport gene

“…Sensitivity to polyoxin D was determined using a liquid assay as described by Cabib (1991). Briefly, cells were grown overnight in YNB supplemented with lysine (30 mg ml-l) and leucine (30 mg ml-l) to a density of approximately 1 x lo7 cells ml-'.…”

“…Previously, peptide conjugates have been used to deliver toxic moieties to C. albicans via the peptide transport system . Synthetic analogues of the polyoxins and nikkomycins, which inhibit chitin synthase (Cabib, 1991), have also been designed to enter C. albicans through the peptide transport system . Hence, these studies may facilitate the design of an effective and specific anticandidal drug by exploiting the concept of ' illicit transport' (Ames e t al., Fickel & Gilvarg, 1973;Hammond et al, 1987;Higgins, 1987) in this opportunistic pathogen.…”

Compounds active against cell walls of medically important fungi

“…In the case of the polyoxins, several derivatives with different amino acid or amino fatty acid compositions were prepared with the hope of improving uptake and biological activity (81,138,144,221,222). With the nikkomycins, the group led by H. Zahner used a different approach in that they first mutagenized the producing strain of Streptomyces tendae and then ran a directed fermentation providing pyrimidines, purines, imidazoles, or amino acids (24,25,66,67,69,101,114 Complicating the issue of differing susceptibilities is a recent report that suggests that, with S. cerevisiae, the gene products of CHSI and CHS2 show differential susceptibilities to the inhibitory effects of polyoxins and nikkomycins, with Chsl being more susceptible (34). As it has been confirmed that several different medically important fungi also have multiple forms of chitin synthetase (26), it is an obvious statement that a determination of the relative importance of each form to the growth of the cell must be made.…”