UNIT 09: Antibiotics active against yeast and fungal cell wall- and lipid biosynthesis

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The usefulness of the brewer/ baker's yeast Saccharomyces cerevisiae in bio-medical research falls under two main headings.

First, the yeast S. cerevisiae shares a close metabolic similarity with pathogenic yeasts/ fungi, e.g., the opportunist pathogen Candida albicans. Metabolic similarities between S. cerevisiae and pathogenic yeasts/ fungi allows one to apply the large S. cerevisiae knowledge base to understanding the biochemistry of the pathogenic yeasts/ fungi. Genomics/ proteomics plays a key role in the process of knowledge base transfer.

Second, S. cerevisiae is classified as a lower eucaryote, which means that mammalian cells share more biochemical characteristics with yeast cells than with bacterial cells, e.g., the terpenoid biosynthetic pathway, which, starting with AcCoA leads to the synthesis of steroids - as we shall discuss today. Yeast cells can be reconstituted with mammalian components by the use of recombinant DNA techniques, to serve as "incubators" for testing/ discovering drugs active in eucaryotes, e.g., seven transmembrane G-protein linked systems.

1. Yeasts and fungi are eucaryotes. They have more in common with animal cells than with bacteria. Thus the ß-lactam antibiotics have no peptidoglycan synthesis to inhibit; however, yeast cells have cell walls made of ß-glucan and chitin that has no counterpart in mammalian cells, which therefore makes them differentially susceptible to chemotherapy. Moreover, there exits significant divergence between eucaryotic cells with respect to the sterol that they synthesize. Many of these sterols are deposited in the cell membrane and this results in differential susceptibility to polyene antibiotics that make pores in membranes that contain sterols.

2. The distinction between eucaryotes and procaryotes is not absolute. The mitochondria of eucaryotic cells are susceptible to antibiotics that selectively inhibit bacterial 70S ribosome function. Even ß-lactam antibiotics find a target in eucaryotic cells despite the absence peptidoglycan synthesis, namely, DNA polymerase alpha.

3. The biochemistry of yeast- and fungal cells differs, nevertheless, from that of other eucaryotic cells in a way that useful selective inhibition is possible in practical situations that are encountered in human and veterinary medicine. Three chemically distinct classes of agents have been found practical application in this connection. Examples of a member of each of the three classes are, (a) miconazole, (b) amphotericin B, and (c) flucytosine, respectively.

4. Eucaryotic cell membranes are organized as phospholipid bilayers and contain additionally sterols as the other major class of lipid components. In animal cells, the sterol is cholesterol, whereas in yeasts and fungi it is ergosterol. This difference can serve as the basis for differential toxicity of two classes of agents, (a) azoles, which inhibit ergosterol synthesis, and (b) polyenes, which disrupt the integrity of ergosterol-containing lipid bilayer membranes but have much less effect on membranes that contain cholesterol, instead. The specificity is not absolute and the toxicity of polyenes is related to their ability to make pores in any membranes that contain sterols.

5. Steps in the conversion of acetate to squalene is common to all organisms, but beginning from squalene the sterol synthesis pathways of animals, plants and fungi diverge. Squalene is converted after several steps to (a) ergosterol, in yeasts and fungi, (b) cholesterol, in animals, and (c) stigmasterol and/or ß-sitosterol, in plants. These sterol end-products become incorporated into fungal-, animal-, and plant cell membranes, respectively.

6. Since the sterol synthesis pathways diverge after squalene, differential inhibition of the synthesis of sterols belonging to the three groups becomes possible. In addition, modification of plant lipids by specific inhibitors can, in turn, affect insects that feed on such treated plants. Sterol synthesis pathways thus provide us with prenylation-specific targets responsive to agents with antifungal-, herbicide-, insecticide-, and hypocholesterolemic effects.

7. An important step in the ergosterol synthesis pathway is the oxidative demethylation at C-14 by lanosterol-14a-methyl demethylase, a cytochrome P-450 oxidase (see outline). Members of the imidazole family, which includes miconazole, ketoconazole, and fluconazole specifically inhibit this step.

What is the structural connection between the azoles and cytochrome P-450 family?

8. The presence of sterols in the plasma membrane sensitizes eucaryotic cells to the action of the polyene antibiotics. Polyene antibiotics associate with membrane sterols in a way that forms circular arrays (see figure sheet). Such circular arrays function as non-specific membrane pores that allow leakage of cell contents.

9. Proteins can associate with membranes by derivitization with:

(a) geranylgeranyl or farnesyl moieties at the C-terminus, and/or

(b) myristoyl or palmitoyl moieties at the N- terminus, functioning as membrane anchors. Geranyl- and farnesyl (generally referred to as "prenyl") moieties come directly off the main line of isoprenoid biosynthesis (see synthesis chart provided). The carboxy terminal tetrapeptide sequence, -CVIM-COOH serves as the recognition sequence for these modifications, which occur on the S-atom of cysteine. The tetrapeptide sequence -CVIM belongs to Ras protein, whose function depends upon being anchored in the membrane. Myristoylation or palmitoylation occurs on the a-carbon of N-terminal glycine.

10. The antibiotic lovastatin inhibits the enzyme hyroxymethyl glutarate CoA reductase which forms mevalonic acid (see chart). Mevalonic acid is used in medical practice to lower serum cholesterol and not in antimicrobial chemotherapy.

11. Mevalonic acid is a precursor of geranyl- and farnesyl pyrophosphate, synthesis of which is also inhibited by lovastatin. As a result, lovastatin acts as a potent inhibitor of protein prenylation and inhibits Ras protein function by preventing its prenylation and consequent anchoring in the cell membrane.

12. Unfortunately for cancer chemotherapy lovastatin cannot be used as an antibiotic for cancer cells that have been transformed by vRas or by a mutant cRas because there are several other proteins present in all cells that are prenylated. As a result, there is little selectivity for inhibition of transformed cells by lovastatin.

13. An alternative to limiting mevalonic acid is to inhibit the activity of the enzyme farnesyl pyrophosphate transferase (FPT) which also must recognize -CVIM as its prenylation substrate. This has been attempted by synthesizing, initially, tetrapeptide analogs of -CVIM, and subsequently peptidomimetic analogs of this sequence.

14. A recent frenzy in peptide research has involved peptidomimetic analogs of CVIM that will selectively inhibit tumors with altered Ras function. The peptide analogs are referred to in the trade as the "CAAX family", i.e., composed of (Cysteine)-(aliphatic amino acid)-(aliphatic amino acid)-(Leucine/ Methionine). The (natural) tetrapeptide, -CVIM, is farnesylated, whereas the analog -CVFM is not, and moreover, CVFM acts as an inhibitor of p21 KB-ras farnsylation, in vitro.

For results with benzodiazepine-based peptidomimetics, see Kohl, N.E. et al. 1993. Selective inhibition of ras-dependent transformation by a farnesyl transferase inhibitor. Science 260: 1934-1942.

15. With the C-terminal peptide analog CVIM as a model target, it is possible to show how one synthesizes a combinatorial library to discover peptide inhibitors of farnesylation presumably based on their ability to function as a decoy of the real sequence.

[graphic illustrations and reprints will be distributed in lecture]

Bacterial signal transduction regulation

Bacterial signal transduction

His-Asp phosphoryl relay systems

How a-helical peptides interact with lipid membranes

How a-helical peptides interact with other a-helical peptides

Enzyme leakage assays

Other modes of b -lactam resistance regulation

b-lactam resistance regulation

AmpC by transcriptional attenuation in S. aureus

AmpC by positive regulation in Citrobacter spp. (Sanders)

Gram positive

Computer tools for studying a-helical peptides

Tools

Protein Predict server

TM Pred server

Hydrophobic cluster analysis (hca) server

PC-TAMMO

		The usefulness of the brewer/ baker's yeast Saccharomyces cerevisiae in bio-medical research falls under two main headings. First, the yeast S. cerevisiae shares a close metabolic similarity with pathogenic yeasts/ fungi, e.g., the opportunist pathogen Candida albicans. Metabolic similarities between S. cerevisiae and pathogenic yeasts/ fungi allows one to apply the large S. cerevisiae knowledge base to understanding the biochemistry of the pathogenic yeasts/ fungi. Genomics/ proteomics plays a key role in the process of knowledge base transfer. Second, S. cerevisiae is classified as a lower eucaryote, which means that mammalian cells share more biochemical characteristics with yeast cells than with bacterial cells, e.g., the terpenoid biosynthetic pathway, which, starting with AcCoA leads to the synthesis of steroids - as we shall discuss today. Yeast cells can be reconstituted with mammalian components by the use of recombinant DNA techniques, to serve as "incubators" for testing/ discovering drugs active in eucaryotes, e.g., seven transmembrane G-protein linked systems. 1. Yeasts and fungi are eucaryotes. They have more in common with animal cells than with bacteria. Thus the ß-lactam antibiotics have no peptidoglycan synthesis to inhibit; however, yeast cells have cell walls made of ß-glucan and chitin that has no counterpart in mammalian cells, which therefore makes them differentially susceptible to chemotherapy. Moreover, there exits significant divergence between eucaryotic cells with respect to the sterol that they synthesize. Many of these sterols are deposited in the cell membrane and this results in differential susceptibility to polyene antibiotics that make pores in membranes that contain sterols. 2. The distinction between eucaryotes and procaryotes is not absolute. The mitochondria of eucaryotic cells are susceptible to antibiotics that selectively inhibit bacterial 70S ribosome function. Even ß-lactam antibiotics find a target in eucaryotic cells despite the absence peptidoglycan synthesis, namely, DNA polymerase alpha. 3. The biochemistry of yeast- and fungal cells differs, nevertheless, from that of other eucaryotic cells in a way that useful selective inhibition is possible in practical situations that are encountered in human and veterinary medicine. Three chemically distinct classes of agents have been found practical application in this connection. Examples of a member of each of the three classes are, (a) miconazole, (b) amphotericin B, and (c) flucytosine, respectively. 4. Eucaryotic cell membranes are organized as phospholipid bilayers and contain additionally sterols as the other major class of lipid components. In animal cells, the sterol is cholesterol, whereas in yeasts and fungi it is ergosterol. This difference can serve as the basis for differential toxicity of two classes of agents, (a) azoles, which inhibit ergosterol synthesis, and (b) polyenes, which disrupt the integrity of ergosterol-containing lipid bilayer membranes but have much less effect on membranes that contain cholesterol, instead. The specificity is not absolute and the toxicity of polyenes is related to their ability to make pores in any membranes that contain sterols. 5. Steps in the conversion of acetate to squalene is common to all organisms, but beginning from squalene the sterol synthesis pathways of animals, plants and fungi diverge. Squalene is converted after several steps to (a) ergosterol, in yeasts and fungi, (b) cholesterol, in animals, and (c) stigmasterol and/or ß-sitosterol, in plants. These sterol end-products become incorporated into fungal-, animal-, and plant cell membranes, respectively. 6. Since the sterol synthesis pathways diverge after squalene, differential inhibition of the synthesis of sterols belonging to the three groups becomes possible. In addition, modification of plant lipids by specific inhibitors can, in turn, affect insects that feed on such treated plants. Sterol synthesis pathways thus provide us with prenylation-specific targets responsive to agents with antifungal-, herbicide-, insecticide-, and hypocholesterolemic effects. 7. An important step in the ergosterol synthesis pathway is the oxidative demethylation at C-14 by lanosterol-14a-methyl demethylase, a cytochrome P-450 oxidase (see outline). Members of the imidazole family, which includes miconazole, ketoconazole, and fluconazole specifically inhibit this step. What is the structural connection between the azoles and cytochrome P-450 family? 8. The presence of sterols in the plasma membrane sensitizes eucaryotic cells to the action of the polyene antibiotics. Polyene antibiotics associate with membrane sterols in a way that forms circular arrays (see figure sheet). Such circular arrays function as non-specific membrane pores that allow leakage of cell contents. 9. Proteins can associate with membranes by derivitization with: (a) geranylgeranyl or farnesyl moieties at the C-terminus, and/or (b) myristoyl or palmitoyl moieties at the N- terminus, functioning as membrane anchors. Geranyl- and farnesyl (generally referred to as "prenyl") moieties come directly off the main line of isoprenoid biosynthesis (see synthesis chart provided). The carboxy terminal tetrapeptide sequence, -CVIM-COOH serves as the recognition sequence for these modifications, which occur on the S-atom of cysteine. The tetrapeptide sequence -CVIM belongs to Ras protein, whose function depends upon being anchored in the membrane. Myristoylation or palmitoylation occurs on the a-carbon of N-terminal glycine. 10. The antibiotic lovastatin inhibits the enzyme hyroxymethyl glutarate CoA reductase which forms mevalonic acid (see chart). Mevalonic acid is used in medical practice to lower serum cholesterol and not in antimicrobial chemotherapy. 11. Mevalonic acid is a precursor of geranyl- and farnesyl pyrophosphate, synthesis of which is also inhibited by lovastatin. As a result, lovastatin acts as a potent inhibitor of protein prenylation and inhibits Ras protein function by preventing its prenylation and consequent anchoring in the cell membrane. 12. Unfortunately for cancer chemotherapy lovastatin cannot be used as an antibiotic for cancer cells that have been transformed by vRas or by a mutant cRas because there are several other proteins present in all cells that are prenylated. As a result, there is little selectivity for inhibition of transformed cells by lovastatin. 13. An alternative to limiting mevalonic acid is to inhibit the activity of the enzyme farnesyl pyrophosphate transferase (FPT) which also must recognize -CVIM as its prenylation substrate. This has been attempted by synthesizing, initially, tetrapeptide analogs of -CVIM, and subsequently peptidomimetic analogs of this sequence. 14. A recent frenzy in peptide research has involved peptidomimetic analogs of CVIM that will selectively inhibit tumors with altered Ras function. The peptide analogs are referred to in the trade as the "CAAX family", i.e., composed of (Cysteine)-(aliphatic amino acid)-(aliphatic amino acid)-(Leucine/ Methionine). The (natural) tetrapeptide, -CVIM, is farnesylated, whereas the analog -CVFM is not, and moreover, CVFM acts as an inhibitor of p21 KB-ras farnsylation, in vitro. For results with benzodiazepine-based peptidomimetics, see Kohl, N.E. et al. 1993. Selective inhibition of ras-dependent transformation by a farnesyl transferase inhibitor. Science 260: 1934-1942. 15. With the C-terminal peptide analog CVIM as a model target, it is possible to show how one synthesizes a combinatorial library to discover peptide inhibitors of farnesylation presumably based on their ability to function as a decoy of the real sequence. [graphic illustrations and reprints will be distributed in lecture]

Bacterial signal transduction regulation		Bacterial signal transduction His-Asp phosphoryl relay systems How a-helical peptides interact with lipid membranes How a-helical peptides interact with other a-helical peptides Enzyme leakage assays
Other modes of b -lactam resistance regulation		b-lactam resistance regulation AmpC by transcriptional attenuation in S. aureus AmpC by positive regulation in Citrobacter spp. (Sanders) Gram positive
Computer tools for studying a-helical peptides		Tools Protein Predict server TM Pred server Hydrophobic cluster analysis (hca) server PC-TAMMO