Chemical Genomics-Based Antifungal Drug Discovery: Targeting Glycosylphosphatidylinositol (GPI) Precursor Biosynthesis
- Paul A. Mann
- ,
- Catherine A. McLellan
- ,
- Sandra Koseoglu
- ,
- Qian Si
- ,
- Elena Kuzmin
- ,
- Amy Flattery
- ,
- Guy Harris
- ,
- Xinwei Sher
- ,
- Nicholas Murgolo
- ,
- Hao Wang
- ,
- Kristine Devito
- ,
- Nuria de Pedro
- ,
- Olga Genilloud
- ,
- Jennifer Nielsen Kahn
- ,
- Bo Jiang
- ,
- Michael Costanzo
- ,
- Charlie Boone
- ,
- Charles G. Garlisi
- ,
- Susan Lindquist
- , and
- Terry Roemer
Abstract
Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.
Note Added After ASAP Publication
This paper was published ASAP on December 12, 2014, with an error in Figure 2. The corrected version reposted with the issue on January 9, 2015.
Results and Discussion
CaFT Screening and GPI Inhibitor Identification
Drug Resistance-Based Mechanism of Action Studies
MB2865 | Ca2323 | Ca1055 | ATCC22019 | MY1396 | ATCC6258 | MY1381 | S288c | MF5668 | ||
---|---|---|---|---|---|---|---|---|---|---|
compd | MOA | S. aureus | C. albicans | C. albicans | C. parapsilosis | C. lusitaniae | C. krusei | C. glabrata | S. cerevisiae | A. fumigatus |
G365 | GWT1 | >128 | 4 (2) | 4 (2) | 2 | >128 | >128 | >128 | >128 | 4 (2) |
G884 | GWT1 | 128 | 4 | 4 | 8 | 64 | 128 | 32 (16) | 16 | 128 (64) |
G642 gepinacin | GWT1 | >128 | 4 (2) | 4 (2) | 2 | 4 | >128 | 4 (2) | 1 (0.5) | (16) |
M720 | MCD4 | >128 | 0.5 (0.25) | 0.5 (0.25) | 0.5 | 0.5 | 1 (0.5) | 0.25 | 1 (0.5) | 0.5 (0.25) |
M743 | MCD4 | >128 | 0.5 | 0.5 (0.25) | 0.5 (0.25) | 1 | 1 (0.5) | 0.5 | 1 (0.5) | 0.25 (0.125) |
AmB | polyene | >128 | 0.25 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
FLZ | azole | >128 | 0.5 (0.25) | >128 | 1 | 1 | 32 | 8 (4) | 4 | >128 |
CAS | candin | >128 | 0.063 | 0.25 | 0.5 | 0.25 | 0.5 | 0.25 (0.125) | 0.25 | (0.016) |
All MIC values are represented as μg/mL.
MIC-50 values (drug concentrations required to inhibit growth 50%) are in parentheses.
Heat map summary of all nonsynonymous mutations identified by Illumina-based NGS (>100× genome coverage) of all independently isolated drug resistant mutants in the pdr5Δ strain, BY4700 (A) or a wild type S288C strain (B). Each column is a summary of all nonsynonymous mutations identified for a particular drug-resistant isolate; no additional nonsynonymous mutations were identified. Red, nonsynonymous mutation that maps to the predicted target Mcd4 (inhibitors M743 and M720) or Gwt1 (G884). Yellow, additional nonsynonymous mutations identified by NGS. Black, no change versus the parental wild type strain gene sequence. Nonsynonymous mutations mapping to the predicted drug target are causal for the drug resistance phenotype as they are faithfully identified in all M720R and M743R isolates from the pdr5Δ strain background as well as the G884R wild type strain background. In several cases, M720R isolates from the wild type starting strain background carry a pdr5 missense mutation (rather than mapping to Mcd4), reflecting a bypass resistance mechanism. Base changes and resulting amino acid substitutions are shown.
Gwt1 Inhibitors Selectively Block Acylation of Yeast Glucosamine Phosphatidylinositol (GlcN-PI) Biosynthesis
GPI Inhibitors Induce the Unfolded Protein Response
Microbiological Evaluation of GPI Inhibitors
GPI Inhibitors Expose Cell Surface β-Glucan and Induce TNFα Secretion
In Vivo Efficacy of M720 in a Murine Infection Model of Candidiasis
Methods
Materials
CaFT Screening and GPI Inhibitor Identification
Microbiological Evaluation of GPI Inhibitors
Measurement of TNFα Secretion
β-Glucan Staining
Mapping of Drug-Resistant Mutants to GPI Targets
Antifungal Efficacy
In Vitro Acylation of Gwt1p Inhibitors
Mammalian Cytotoxicity Assessment
Supporting Information
The following file is available free of charge on the ACS Publications website at DOI: 10.1021/id5000212.
Tables S1–S4 contain general strain information, mammalian cytotoxicity results, and expanded bacterial spectrum testing; Figures S1–S7 contain additional data describing CaFT analysis, heterozygote spot testing, Aspergillus zones of inhibition, time–kill assays, conditional mutant response, and Mcd4 inhibitor stability in plasma (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgment
We thank Carl Balibar and Juliana Malinverni for critically reading the manuscript and Luke Whitesell for technical assistance. This work was supported in part by a grant from Genome Canada and Genome Quebec.
CaFT | C. albicans fitness test |
GPI | glucosylphosphatidylinositol |
MOA | mechanism of action |
EtNP | ethanolamine phosphate |
Man1 | mannose 1 |
NGS | next-generation sequencing |
TLRs | Toll-like receptors |
COPI | coat protein complex I |
MIC | minimal inhibitor concentration |
GlcN-PI | glucosamine phosphatidylinositol |
UPR | unfolded protein response |
AmB | amphotericin B |
FLZ | fluconazole |
CSP | caspofungin |
FIC | fractional inhibitory concentrations |
SOC | standard of care |
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- 14Richard, M. L. and Plaine, A. (2007) Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicans Eukaryot. Cell. 6, 119– 133 DOI: 10.1128/EC.00297-06Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXitVersbc%253D&md5=afa6c59bf87ddae8c48facae2229da07Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicansRichard, Mathias L.; Plaine, ArmelEukaryotic Cell (2007), 6 (2), 119-133CODEN: ECUEA2; ISSN:1535-9778. (American Society for Microbiology)A review that focuses on the function of glycosylphosphatidylinositol-anchored proteins in Candida albicans.
- 15Pittet, M. and Conzelmann, A. (2007) Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae Biochim. Biophys. Acta 1771, 405– 420 DOI: 10.1016/j.bbalip.2006.05.015Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjsVeru70%253D&md5=eb0ba642fce1f308d70f85d9458748fdBiosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiaePittet, Martine; Conzelmann, AndreasBiochimica et Biophysica Acta, Molecular and Cell Biology of Lipids (2007), 1771 (3), 405-420CODEN: BBMLFG; ISSN:1388-1981. (Elsevier Ltd.)A review. Like most other eukaryotes, Saccharomyces cerevisiae harbors a GPI anchoring machinery and uses it to attach proteins to membranes. While a few GPI proteins reside permanently at the plasma membrane, a majority of them gets further processed and is integrated into the cell wall by a covalent attachment to cell wall glucans. The GPI biosynthetic pathway is necessary for growth and survival of yeast cells. The GPI lipids are synthesized in the ER and added onto proteins by a pathway comprising 12 steps, carried out by 23 gene products, 19 of which are essential. Some of the estd. 60 GPI proteins predicted from the genome sequence serve enzymic functions required for the biosynthesis and the continuous shape adaptations of the cell wall, others seem to be structural elements of the cell wall and yet others mediate cell adhesion. Because of its genetic tractability S. cerevisiae is an attractive model organism not only for studying GPI biosynthesis in general, but equally for investigating the intracellular transport of GPI proteins and the peculiar role of GPI anchoring in the elaboration of fungal cell walls.
- 16Klis, F. M., Sosinska, G. J., de Groot, P. W., and Brul, S. (2009) Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence FEMS Yeast Res. 9, 1013– 1028 DOI: 10.1111/j.1567-1364.2009.00541.xGoogle Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlOis7bK&md5=03afa73bf2dfb2456c3420b9aa94c729Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulenceKlis, Frans M.; Sosinska, Grazyna J.; de Groot, Piet W. J.; Brul, StanleyFEMS Yeast Research (2009), 9 (7), 1013-1028CODEN: FYREAG; ISSN:1567-1356. (Wiley-Blackwell)A review. The cell wall of Candida albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, contg. c. 20 different protein species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat proteins vary widely in function. Many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell-cell aggregation. They further include iron acquisition proteins, superoxide dismutases, and yapsin-like aspartic proteases. In addn., several covalently linked carbohydrate-active enzymes are present, whose precise functions remain hitherto largely elusive. The expression levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary enormously. They depend on the mode of growth and the combined inputs of several signaling pathways that sense environmental conditions. This is reflected in the unusually long intergenic regions of most of these genes. Finally, the precise location of several covalently linked CWPs is temporally and spatially regulated. It is concluded that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence and that their expression is tightly controlled.
- 17Orlean, P. and Menon, A. K. (2007) Thematic review series: Lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids J. Lipid Res. 48, 993– 1011 DOI: 10.1194/jlr.R700002-JLR200Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXls1ejtLo%253D&md5=cdba29bd6397497683c83925fb816ca5GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipidsOrlean, Peter; Menon, Anant K.Journal of Lipid Research (2007), 48 (5), 993-1011CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology, Inc.)A review. Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that were translocated across the ER membrane nd that display a GPI signal anchor sequence pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but 1 of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, the authors describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.
- 18Sütterlin, C., Horvath, A., Gerold, P., Schwarz, R. T., Wang, Y., Dreyfuss, M., and Riezman, H. (1997) Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis EMBO J. 16, 6374– 6383 DOI: 10.1093/emboj/16.21.6374Google ScholarThere is no corresponding record for this reference.
- 19Wang, Y., Dreyfuss, M., Ponelle, M., Oberer, L., and Riezman, H. A Glycosylphosphatidylinositol-anchoring inhibitor with an unusual tetracarboxocyclic sesterpene skeleton from the fungus Codinaea simplex. Tetrahedron 54, 6415– 6426. DOI: 10.1016/S0040-4020(98)00322-6Google ScholarThere is no corresponding record for this reference.
- 20Hong, Y., Maeda, Y., Watanabe, R., Ohishi, K., Mishkind, M., Riezman, H., and Kinoshita, T. (1999) Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol J. Biol. Chem. 274, 35099– 35106 DOI: 10.1074/jbc.274.49.35099Google ScholarThere is no corresponding record for this reference.
- 21Wiedman, J. M., Fabre, A. L., Taron, B. W., Taron, C. H., and Orlean, P. (2007) In vivo characterization of the GPI assembly defect in yeast mcd4–174 mutants and bypass of the Mcd4p-dependent step in mcd4Delta cells FEMS Yeast Res. 7, 78– 83 DOI: 10.1111/j.1567-1364.2006.00139.xGoogle Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhslyhuro%253D&md5=be87a6b02cd4021df9acfca6ebd728d4In vivo characterization of the GPI assembly defect in yeast mcd4-174 mutants and bypass of the Mcd4p-dependent step in mcd4Δ cellsWiedman, Jill M.; Fabre, Anne-Lise; Taron, Barbara W.; Taron, Christopher H.; Orlean, PeterFEMS Yeast Research (2007), 7 (1), 78-83CODEN: FYREAG; ISSN:1567-1356. (Blackwell Publishing Ltd.)Yeast mcd4-174 mutants are blocked in glycosylphosphatidylinositol (GPI) anchoring of protein, but the stage at which GPI biosynthesis is interrupted in vivo has not been identified, and Mcd4p has also been implicated in phosphatidylserine and ATP transport. The authors report that the major GPI that accumulates in mcd4-174 in vivo is Man2-GlcN-(acyl-Ins)PI, consistent with proposals that Mcd4p adds phosphoethanolamine to the 1st mannose of yeast GPI precursors. Mcd4p-dependent modification of GPIs can partially be bypassed in the mcd4-174/gpi11 double mutant and in mcd4Δ mutants by high-level expression of PIG-B and GPI10, which resp. encode the human and yeast mannosyltransferases that add the 3rd mannose of the GPI precursor. Rescue of mcd4Δ by GPI10 indicates that Mcd4p-dependent addn. of EthN-P to the 1st mannose of GPIs is not obligatory for transfer of the 3rd mannose by Gpi10p.
- 22Tsukahara, K., Hata, K., Nakamoto, K., Sagane, K., Watanabe, N. A., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly Mol. Microbiol. 48, 1029– 1042 DOI: 10.1046/j.1365-2958.2003.03481.xGoogle Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktV2gsL4%253D&md5=1049da3e97c3df636219d903cc2f939cMedicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assemblyTsukahara, Kappei; Hata, Katsura; Nakamoto, Kazutaka; Sagane, Koji; Watanabe, Nao-aki; Kuromitsu, Junro; Kai, Junko; Tsuchiya, Mamiko; Ohba, Fuminori; Jigami, Yoshifumi; Yoshimatsu, Kentaro; Nagasu, TakeshiMolecular Microbiology (2003), 48 (4), 1029-1042CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)Glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small mol. inhibitors of the cell surface presentation of GPI-anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI-anchored cell wall mannoproteins. By means of a yeast cell-based screening procedure, we discovered a compd., 1-[4-butylbenzyl]isoquinoline (I), that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compd. resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage-dependent suppressor of the I-induced phenotypes. GWT1 knock-out cells showed similar phenotypes to I-treated wild-type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to I each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compd.
- 23Gaynor, E. C., Mondésert, G., Grimme, S. J., Reed, S. I., Orlean, P., and Emr, S. D. (1999) MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast Mol. Biol. Cell 10, 627– 648 DOI: 10.1091/mbc.10.3.627Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvVyku7c%253D&md5=cc36193454987edf3eea47f0c292fa83MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeastGaynor, Erin C.; Mondesert, Guillaume; Grimme, Stephen J.; Reed, Steve I.; Orlean, Peter; Emr, Scott D.Molecular Biology of the Cell (1999), 10 (3), 627-648CODEN: MBCEEV; ISSN:1059-1524. (American Society for Cell Biology)Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a crit. role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temp.-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphol. defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. Mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.
- 24Dennehy, K. M. and Brown, G. D. (2007) The role of the beta-glucan receptor dectin-1 in control of fungal infection J. Leukoc. Biol. 82, 253– 258 DOI: 10.1189/jlb.1206753Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos12rtbk%253D&md5=369bd2e12247f8cc55c6a65bfaa63432The role of the β-glucan receptor Dectin-1 in control of fungal infectionDennehy, Kevin M.; Brown, Gordon D.Journal of Leukocyte Biology (2007), 82 (2), 253-258CODEN: JLBIE7; ISSN:0741-5400. (Federation of American Societies for Experimental Biology)A review. During fungal infection, a variety of receptors initiates immune responses, including TLR and the β-glucan receptor Dectin-1. TLR recognition of fungal ligands and subsequent signaling through the MyD88 pathway were thought to be the most important interactions required for the control of fungal infection. However, recent papers have challenged this view, highlighting the role of Dectin-1 in induction of cytokine responses and the respiratory burst. Two papers, using independently derived, Dectin-1-deficient mice, address the role of Dectin-1 in control of fungal infection. Saijo et al. (Nat. Immunol., 2007, 8, 39-46) argue that Dectin-1 plays a minor role in control of Pneumocystis carinii by direct killing and that TLR-mediated cytokine prodn. controls P. carinii and Candida albicans. By contrast, Taylor et al. (Nat. Immunol, 2007, 8, 31-38) argue that Dectin-1-mediated cytokine and chemokine prodn., leading to efficient recruitment of inflammatory cells, is required for control of fungal infection. Here, the authors argue that collaborative responses induced during infection may partially explain these apparently contradictory results. They propose that Dectin-1 is the first of many pattern recognition receptors that can mediate their own signaling, as well as synergize with TLR to initiate specific responses to infectious agents.
- 25Umemura, M., Okamoto, M., Nakayama, K., Sagane, K., Tsukahara, K., Hata, K., and Jigami, Y. (2003) GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast J. Biol. Chem. 278, 23639– 23647 DOI: 10.1074/jbc.M301044200Google ScholarThere is no corresponding record for this reference.
- 26Watanabe, N. A., Miyazaki, M., Horii, T., Sagane, K., Tsukahara, K., and Hata, K. (2012) E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis Antimicrob. Agents Chemother. 56, 960– 971 DOI: 10.1128/AAC.00731-11Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Gruro%253D&md5=a489c9d19296eaa8b65513de5b133ce3E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesisWatanabe, Nao-aki; Miyazaki, Mamiko; Horii, Takaaki; Sagane, Koji; Tsukahara, Kappei; Hata, KatsuraAntimicrobial Agents and Chemotherapy (2012), 56 (2), 960-971CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concns. (IC50s) of 0.3 to 0.6 μM but had no inhibitory activity against human Pig-Wp even at concns. as high as 100 μM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude ext. and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concns. above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.
- 27McLellan, C. A., Whitesell, L., King, O. D., Lancaster, A. K., Mazitschek, R., and Lindquist, S. (2012) Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity ACS Chem. Biol. 7, 1520– 1528 DOI: 10.1021/cb300235mGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovFaktr0%253D&md5=f5d23f76cbc1942730dce3af6b2af115Inhibiting GPI Anchor Biosynthesis in Fungi Stresses the Endoplasmic Reticulum and Enhances ImmunogenicityMcLellan, Catherine A.; Whitesell, Luke; King, Oliver D.; Lancaster, Alex K.; Mazitschek, Ralph; Lindquist, SusanACS Chemical Biology (2012), 7 (9), 1520-1528CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biol. of GPI-anchor synthesis is difficult, esp. in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, the discovery of a new small mol. christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a crit. acyltransferase required for the biosynthesis of fungal GPI anchors, is reported. After delineating the target specificity of gepinacin using genetic and biochem. techniques, it was used to probe key, therapeutically relevant consequences of disrupting GPI anchor metab. in fungi. It was found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compd. results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture expts. to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concns. impaired filamentation and unmasked cell wall β-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepinacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi.
- 28Lee, M. C., Miller, E. A., Goldberg, J., Orci, L., and Schekman, R. (2004) Bi-directional protein transport between the ER and Golgi Annu. Rev. Cell Dev. Biol. 20, 87– 123 DOI: 10.1146/annurev.cellbio.20.010403.105307Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVaqu7bM&md5=ae02d65edd306dafa84ef899b1d628cdBi-directional protein transport between the ER and GolgiLee, Marcus C. S.; Miller, Elizabeth A.; Goldberg, Jonathan; Orci, Lelio; Schekman, RandyAnnual Review of Cell and Developmental Biology (2004), 20 (), 87-123CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)A review. The endoplasmic reticulum (ER) and the Golgi app. comprise the 1st 2 steps in protein secretion. Vesicular carriers mediate a continuous flux of proteins and lipids between these compartments, reflecting the transport of newly synthesized proteins out of the ER and the retrieval of escaped ER residents and vesicle machinery. Anterograde and retrograde transport is mediated by distinct sets of cytosolic coat proteins, the COPII and COPI coats, resp., which act on the membrane to capture cargo proteins into nascent vesicles. Here, the authors review the mechanisms that govern coat recruitment to the membrane, cargo capture into a transport vesicle, and accurate delivery to the target organelle.
- 29Kodera, C., Yorimitsu, T., Nakano, A., and Sato, K. (2011) Sed4p stimulates Sar1p GTP hydrolysis and promotes limited coat disassembly Traffic 12, 591– 599 DOI: 10.1111/j.1600-0854.2011.01173.xGoogle ScholarThere is no corresponding record for this reference.
- 30Letourneur, F., Gaynor, E. C., Hennecke, S., Demolliere, C., Duden, R., Emr, S. D., Riezman, H., and Cosson, P. (1994) Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79, 1199– 1207 DOI: 10.1016/0092-8674(94)90011-6Google ScholarThere is no corresponding record for this reference.
- 31Ma, W. and Goldberg, J. (2013) Rules for the recognition of dilysine retrieval motifs by coatomer EMBO J. 32, 926– 937 DOI: 10.1038/emboj.2013.41Google ScholarThere is no corresponding record for this reference.
- 32Carvajal, E., van den Hazel, H. B., Cybularz-Kolaczkowska, A., Balzi, E., and Goffeau, A. (1997) Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes Mol. Gen Genet. 256, 406– 415 DOI: 10.1007/s004380050584Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhsl2juw%253D%253D&md5=fb7163231216cf3d46323b51f5490edeMolecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genesCarvajal, E.; Van Den Hazel, H. B.; Cybularz-Kolaczkowska, A.; Balzi, E.; Goffeau, A.Molecular & General Genetics (1997), 256 (4), 406-415CODEN: MGGEAE; ISSN:0026-8925. (Springer-Verlag)Mutations at the yeast PDR1 transcriptional regulator locus are responsible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, assocd. with the appearance of multiple drug resistance. The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidrug resistance mutants, 1 intragenic suppressor and 6 wild types, have been detd. Single amino acid substitutions were shown to result from the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1-7 (P298A) and pdr1-8 (L1036W), whereas the intragenic suppressor mutant pdr1-100 is deleted for the two amino acids L537 and A538. An isogenic series of strains was constructed contg. the mutant alleles pdr1-3, pdr1-6 and pdr1-8 integrated into the genome. It was found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N-oxide and ketoconazole were increased in all three mutants. The increase was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mutants. Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter and the pdr1-3 mutation resulted in the highest level of promoter induction. Concomitantly. the level of PDR5 mRNA, of Pdr5p protein, and of its assocd. nucleoside triphosphatase activity, was strongly increased in the plasma membranes of the PDR1 mutants. Again, the pdr1-3 allele was assocd. with a stronger effect than the pdr1-8 and pdr1-6 alleles. The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p transcription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency. These gain-of-function mutations support the concept, recently proposed, that in members of the large family of yeast Zn2Cys6 transcription factors a central inhibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mutations). This domain may interact in a locked conformation with a putative, more C-terminally located inhibitory domain (mutated in pdr1-3), and with the putative activation domain (mutated in pdr1-8).
- 33Sagane, K., Umemura, M., Ogawa-Mitsuhashi, K., Tsukahara, K., Yoko-o, T., and Jigami, Y. (2011) Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p J. Biol. Chem. 286, 14649– 14658 DOI: 10.1074/jbc.M110.193490Google ScholarThere is no corresponding record for this reference.
- 34Zhu, Y., Vionnet, C., and Conzelmann, A. (2006) Ethanolaminephosphate side chain added to glycosylphosphatidylinositol (GPI) anchor by mcd4p is required for ceramide remodeling and forward transport of GPI proteins from endoplasmic reticulum to Golgi J. Biol. Chem. 281, 19830– 19839 DOI: 10.1074/jbc.M601425200Google ScholarThere is no corresponding record for this reference.
- 35Miyazaki, M., Horii, T., Hata, K., Watanabe, N. A., Nakamoto, K., Tanaka, K., Shirotori, S., Murai, N., Inoue, S., Matsukura, M., Abe, S., Yoshimatsu, K., and Asada, M. (2011) In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds Antimicrob. Agents Chemother. 55, 4652– 4658 DOI: 10.1128/AAC.00291-11Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2nsL%252FP&md5=cb380963b1f986ed2e0d72ed5067e19dIn vitro activity of E1210, a novel antifungal, against clinically important yeasts and moldsMiyazaki, Mamiko; Horii, Takaaki; Hata, Katsura; Watanabe, Nao-aki; Nakamoto, Kazutaka; Tanaka, Keigo; Shirotori, Syuji; Murai, Norio; Inoue, Satoshi; Matsukura, Masayuki; Abe, Shinya; Yoshimatsu, Kentaro; Asada, MakotoAntimicrobial Agents and Chemotherapy (2011), 55 (10), 4652-4658CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)E1210 is a new antifungal compd. with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clin. fungal isolates. E1210 showed potent activities against most Candida spp. (MIC90 of ≤0.008 to 0.06 μg/mL), except for Candida krusei (MICs of 2 to >32 μg/mL). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC90 of 0.13 μg/mL). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 μg/mL), Scedosporium prolificans (MIC of 0.03 μg/mL), and Paecilomyces lilacinus (MICs of 0.06 μg/mL) among the compds. tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been obsd. with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.
- 36Poulain, D. and Jouault, T. (2004) Candida albicans cell wall glycans, host receptors and responses: elements for a decisive crosstalk Curr. Opin. Microbiol. 7, 342– 349 DOI: 10.1016/j.mib.2004.06.011Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmt1Omsbg%253D&md5=f563b4eb9e95bb8616d8e58dc707d8c1Candida albicans cell wall glycans, host receptors and responses: elements for a decisive crosstalkPoulain, Daniel; Jouault, ThierryCurrent Opinion in Microbiology (2004), 7 (4), 342-349CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)A review. Candida albicans has adapted to live on the mucosal surfaces of animals. The human species has accepted it. By contrast to numerous other commensals, C. albicans has a prominent ability to invade virtually all tissues of a host presenting with natural or acquired defects in homeostasis. C. albicans uses considerable energy to synthesize glycans, which are present either as polymers or as glyconjugates. These glycan mols. play a prominent role in the biol. of C. albicans by controlling the structure and plasticity of the cell wall, and are also involved in yeast-host interactions. These glycans are recognized as non-self by host innate and adaptative immunity. The signal they induce in the host depends on the glycan code, which is detd. by the nature of the sugar, the anomer type of linkage and branching, and the length of the oligosaccharide chains. However, this model is not static because the nature of the C. albicans mol. carrying such glycan codes and their expression at the cell wall surface also dets. the host response, and, in turn, the regulation of cell wall glycan arrangement dynamics in C. albicans depends on host stimuli. Candida glycans therefore play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and resistance and infection. A goal of current research concerning the virulence attributes of C. albicans will be to det. to what extent this species is able to regulate its glycan code as a response to the host.
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- 39Kushida, H., Nakajima, S., Uchiyama, S., Nagashima, M., Kojiri, K., Kawamura, K., and Suda, H. (1999) Antifungal substances BE-49385 and process for their production. U.S. Patent 5,928,910.Google ScholarThere is no corresponding record for this reference.
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- 41Sütterlin, C., Escribano, M. V., Gerold, P., Maeda, Y., Mazon, M. J., Kinoshita, T., Schwarz, R. T., and Riezman, H. (1998) Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis Biochem. J. 332, 153– 159Google ScholarThere is no corresponding record for this reference.
- 42McConville, M. J. and ad Ferguson, M. A. J. (1993) The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes Biochem. J. 294, 305– 324Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlvF2hs70%253D&md5=23f9cf6089c12012eaa42693daafc790The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotesMcConville, Malcolm J.; Ferguson, Michael A. J.Biochemical Journal (1993), 294 (2), 305-24CODEN: BIJOAK; ISSN:0264-6021.A review with >200 refs. on the structure, function and biosynthesis of glycosyl-phosphatidylinositol (GPI) anchors in the title organisms. There may be significant differences in the function of GPI protein anchors in unicellular vs. metazoan organisms. Some parasitic protozoa synthesize exotic GPI-related structures which are not attached to proteins. Evolutionary aspects of the GPI family are also discussed.
- 43Hata, K., Horii, T., Miyazaki, M., Watanabe, N. A., Okubo, M., Sonoda, J., Nakamoto, K., Tanaka, K., Shirotori, S., Murai, N., Inoue, S., Matsukura, M., Abe, S., Yoshimatsu, K., and Asada, M. (2011) Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis Antimicrob. Agents Chemother. 55, 4543– 4551 DOI: 10.1128/AAC.00366-11Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2ns7fE&md5=ff015a73a4108a15fe6474a7411a6044Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosisHata, Katsura; Horii, Takaaki; Miyazaki, Mamiko; Watanabe, Nao-aki; Okubo, Miyuki; Sonoda, Jiro; Nakamoto, Kazutaka; Tanaka, Keigo; Shirotori, Syuji; Murai, Norio; Inoue, Satoshi; Matsukura, Masayuki; Abe, Shinya; Yoshimatsu, Kentaro; Asada, MakotoAntimicrobial Agents and Chemotherapy (2011), 55 (10), 4543-4551CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)E1210 is a 1st-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and ref. antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater redn. in the no. of oral CFU than the control treatment. In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice. E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice. E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to det. E1210's potential for the treatment of disseminated fungal infections are indicated.
- 44Lee, A. Y., St. Onge, R. P., Proctor, M. J., Wallace, I. M., Nile, A. H., Spagnuolo, P. A., Jitkova, Y., Gronda, M., Wu, Y., Kim, M. K., Cheung-Ong, K., Torres, N. P., Spear, E. D., Han, M. K., Schlecht, U., Suresh, S., Duby, G., Heisler, L. E., Surendra, A., Fung, E., Urbanus, M. L., Gebbia, M., Lissina, E., Miranda, M., Chiang, J. H., Aparicio, A. M., Zeghouf, M., Davis, R. W., Cherfils, J., Boutry, M., Kaiser, C. A., Cummins, C. L., Trimble, W. S., Brown, G. W., Schimmer, A. D., Bankaitis, V. A., Nislow, C., Bader, G. D., and Giaever, G. (2014) Mapping the cellular response to small molecules using chemogenomic fitness signatures Science 344, 208– 211 DOI: 10.1126/science.1250217Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlslSmtbw%253D&md5=543c8d40d54c94f789521ed29859d403Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness SignaturesLee, Anna Y.; St. Onge, Robert P.; Proctor, Michael J.; Wallace, Iain M.; Nile, Aaron H.; Spagnuolo, Paul A.; Jitkova, Yulia; Gronda, Marcela; Wu, Yan; Kim, Moshe K.; Cheung-Ong, Kahlin; Torres, Nikko P.; Spear, Eric D.; Han, Mitchell K. L.; Schlecht, Ulrich; Suresh, Sundari; Duby, Geoffrey; Heisler, Lawrence E.; Surendra, Anuradha; Fung, Eula; Urbanus, Malene L.; Gebbia, Marinella; Lissina, Elena; Miranda, Molly; Chiang, Jennifer H.; Aparicio, Ana Maria; Zeghouf, Mahel; Davis, Ronald W.; Cherfils, Jacqueline; Boutry, Marc; Kaiser, Chris A.; Cummins, Carolyn L.; Trimble, William S.; Brown, Grant W.; Schimmer, Aaron D.; Bankaitis, Vytas A.; Nislow, Corey; Bader, Gary D.; Giaever, GuriScience (Washington, DC, United States) (2014), 344 (6180), 208-211CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small mols. affects biol. and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compd. in vivo to profile 3250 small mols. in a systematic and unbiased manner. We identified 317 compds. that specifically perturb the function of 121 genes and characterized the mechanism of specific compds. Global anal. revealed that the cellular response to small mols. is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chems., and biol. processes.
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References
ARTICLE SECTIONSThis article references 44 other publications.
- 1Pfaller, M. A. and Diekema, D. J. (2007) Epidemiology of invasive candidiasis: a persistent public health problem Clin. Microbiol. Rev. 20, 133– 163 DOI: 10.1128/CMR.00029-061https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhvFKnur0%253D&md5=e0278d710f17b1fcceca25450a884c19Epidemiology of invasive candidiasis: a persistent public health problemPfaller, M. A.; Diekema, D. J.Clinical Microbiology Reviews (2007), 20 (1), 133-163CODEN: CMIREX; ISSN:0893-8512. (American Society for Microbiology)A review. The contemporary epidemiol. of invasive candidiasis is discussed. Trends in incidence, mortality, species distribution, and antifungal resistance are described. Issues of cost and emerging strategies for the treatment and prevention of this important invasive mycosis are also addressed.
- 2Brown, G. D., Denning, D. W., Gow, N. A., Levitz, S. M., Netea, M. G., and White, T. C. (2012) Hidden killers: human fungal infections Sci. Transl. Med. 4, 165rv13 DOI: 10.1126/scitranslmed.3004404There is no corresponding record for this reference.
- 3Ostrosky-Zeichner, L., Casadevall, A., Galgiani, J. N., Odds, F. C., and Rex, J. H. (2010) An insight into the antifungal pipeline: selected new molecules and beyond Nat. Rev. Drug Discovery 9, 719– 727 DOI: 10.1038/nrd30743https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtVCmurvK&md5=d7ccb58b653d567179e53437061df80eAn insight into the antifungal pipeline: selected new molecules and beyondOstrosky-Zeichner, Luis; Casadevall, Arturo; Galgiani, John N.; Odds, Frank C.; Rex, John H.Nature Reviews Drug Discovery (2010), 9 (9), 719-727CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)A review. Invasive fungal infections are increasing in incidence and are assocd. with substantial mortality. Improved diagnostics and the availability of new antifungals have revolutionized the field of medical mycol. in the past decades. This Review focuses on recent developments in the antifungal pipeline, concg. on promising candidates such as new azoles, polyenes and echinocandins, as well as agents such as nikkomycin Z and the sordarins. Developments in vaccines and antibody-based immunotherapy are also discussed. Few therapeutic products are currently in active development, and progression of therapeutic agents with fungus-specific mechanisms of action is of key importance.
- 4Roemer, T. and Krysan, D. J. Antifungal drug development: challenges, unmet clinical needs, and new approaches. (2014) Cold Spring Harb Perspect. Med. 4 (5), DOI: 10.1101/cshperspect.a019703 .There is no corresponding record for this reference.
- 5Roemer, T., Jiang, B., Davison, J., Ketela, T., Veillette, K., Breton, A., Tandia, F., Linteau, A., Sillaots, S., Marta, C., Martel, N., Veronneau, S., Lemieux, S., Kauffman, S., Becker, J., Storms, R., Boone, C., and Bussey, H. (2003) Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery Mol. Microbiol. 50, 167– 181 DOI: 10.1046/j.1365-2958.2003.03697.x5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXotFOhur0%253D&md5=4c1ba1bcd6ce3a32f36a89a7f5c8f4fbLarge-scale essential gene identification in Candida albicans and applications to antifungal drug discoveryRoemer, Terry; Jiang, Bo; Davison, John; Ketela, Troy; Veillette, Karynn; Breton, Anouk; Tandia, Fatou; Linteau, Annie; Sillaots, Susan; Marta, Catarina; Martel, Nick; Veronneau, Steeve; Lemieux, Sebastien; Kauffman, Sarah; Becker, Jeff; Storms, Reginald; Boone, Charles; Bussey, HowardMolecular Microbiology (2003), 50 (1), 167-181CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)C. albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections, antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACE (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologs of the Saccharomyces cerevisiae essential gene set by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examn. of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal vs. static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addn., the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their resp. conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.
- 6Xu, D., Jiang, B., Ketela, T., Lemieux, S., Veillette, K., Martel, N., Davison, J., Sillaots, S., Trosok, S., Bachewich, C., Bussey, H., Youngman, P., and Roemer, T. (2007) Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans PLoS Pathog. 3, e92 DOI: 10.1371/journal.ppat.0030092There is no corresponding record for this reference.
- 7Roemer, T., Xu, D., Singh, S. B., Parish, C. A., Harris, G., Wang, H., Davies, J. E., and Bills, G. F. (2011) Confronting the challenges of natural product-based antifungal discovery Chem. Biol. 18, 148– 164 DOI: 10.1016/j.chembiol.2011.01.0097https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXit1Kjtro%253D&md5=7fe45a7b27bbcf2f222631036b072e76Confronting the Challenges of Natural Product-Based Antifungal DiscoveryRoemer, Terry; Xu, Deming; Singh, Sheo B.; Parish, Craig A.; Harris, Guy; Wang, Hao; Davies, Julian E.; Bills, Gerald F.Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (2), 148-164CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)A review. Starting with the discovery of penicillin, the pharmaceutical industry has relied extensively on natural products (NPs) as an unparalleled source of bioactive small mols. suitable for antibiotic development. However, the discovery of structurally novel and chem. tractable NPs with suitable pharmacol. properties as antibiotic leads has waned in recent decades. Today, the repetitive "rediscovery" of previously known NP classes with limited antibiotic lead potential dominates most industrial efforts. This limited productivity, exacerbated by the significant financial and resource requirements of such activities, has led to a broad de-emphasis of NP research by most pharmaceutical companies, including most recently Merck. Here we review our strategies-both technol. and philosophical-in addressing current antifungal discovery bottlenecks in target identification and validation and how such efforts may improve NP-based antimicrobial discoveries when aligned with NP screening and dereplication.
- 8Shoemaker, D. D., Lashkari, D. A., Morris, D., Mittmann, M., and Davis, R. W. (1996) Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy Nat. Genet. 14, 450– 456 DOI: 10.1038/ng1296-4508https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XntlGgt7k%253D&md5=078e8c61adac5e44de40fe221730d2d7Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategyShoemaker, Daniel D.; Lashkari, Deval A.; Morris, Don; Mittmann, Mike; Davis, Ronald W.Nature Genetics (1996), 14 (4), 450-456CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Co.)A quant. and highly parallel method for analyzing deletion mutants has been developed to aid in detg. the biol. function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae. This approach uses a PCR targeting strategy to generate large nos. of deletion strains. Each deletion strain is labeled with a unique 20-base tag sequence that can be detected by hybridization to a high-d. oligonucleotide array. The tags serve as unique identifiers (mol. bar codes) that allow anal. of large nos. of deletion strains simultaneously through selective growth conditions. Hybridization expts. show that the arrays are specific, sensitive and quant. A pilot study with 11 known yeast genes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome anal. with a single selection growth condition and a single hybridization.
- 9Giaever, G., Shoemaker, D. D., Jones, T. W., Liang, H., Winzeler, E. A., Astromoff, A., and Davis, R. W. (1999) Genomic profiling of drug sensitivities via induced haploinsufficiency Nat. Genet. 21, 278– 283 DOI: 10.1038/67919https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXitVCitL0%253D&md5=2a1d1ef187aed84b3499e53af5baada4Genomic profiling of drug sensitivities via induced haploinsufficiencyGiaever, Guri; Shoemaker, Daniel D.; Jones, Ted W.; Liang, Hong; Winzeler, Elizabeth A.; Astromoff, Anna; Davis, Ronald W.Nature Genetics (1999), 21 (3), 278-283CODEN: NGENEC; ISSN:1061-4036. (Nature America)Lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any drug that acts on the product of this gene. This haploinsufficient phenotype thereby identifies the gene product of the heterozygous locus as the likely drug target. We exploited this finding in a genomic approach to drug-target identification. Genome sequence information was used to generate molecularly tagged heterozygous yeast strains that were pooled, grown competitively in drug and analyzed for drug sensitivity using high-d. oligonucleotide arrays. Individual heterozygous strain anal. verified six known drug targets. Parallel anal. identified the known target and two hypersensitive loci in a mixed culture of 233 strains in the presence of the drug tunicamycin. Our discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may have important consequences in pharmacogenomics and variable drug toxicity obsd. in human populations.
- 10Jiang, B., Xu, D., Allocco, J., Parish, C., Davison, J., Veillette, K., Sillaots, S., Hu, W., Rodriguez-Suarez, R., Trosok, S., Zhang, L., Li, Y., Rahkhoodaee, F., Ransom, T., Martel, N., Wang, H., Gauvin, D., Wiltsie, J., Wisniewski, D., Salowe, S., Kahn, J. N., Hsu, M. J., Giacobbe, R., Abruzzo, G., Flattery, A., Gill, C., Youngman, P., Wilson, K., Bills, G., Platas, G., Pelaez, F., Diez, M. T., Kauffman, S., Becker, J., Harris, G., Liberator, P., and Roemer, T. (2008) PAP inhibitor with in vivo efficacy identified by Candida albicans genetic profiling of natural products Chem. Biol. 15, 363– 374 DOI: 10.1016/j.chembiol.2008.02.01610https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXkvFWqt7w%253D&md5=a092e9c8eb95781e38397709bf005ca7PAP inhibitor with in vivo efficacy identified by Candida albicans genetic profiling of natural productsJiang, Bo; Xu, Deming; Allocco, John; Parish, Craig; Davison, John; Veillette, Karynn; Sillaots, Susan; Hu, Wenqi; Rodriguez-Suarez, Roberto; Trosok, Steve; Zhang, Li; Li, Yang; Rahkhoodaee, Fariba; Ransom, Tara; Martel, Nick; Wang, Hao; Gauvin, Daniel; Wiltsie, Judyann; Wisniewski, Douglas; Salowe, Scott; Nielsen-Kahn, Jennifer; Hsu, Ming-Jo; Giacobbe, Robert; Abruzzo, George; Flattery, Amy; Gill, Charles; Youngman, Phil; Wilson, Ken; Bills, Gerald; Platas, Gonzalo; Pelaez, Fernando; Diez, Maria Teresa; Kauffman, Sarah; Becker, Jeff; Harris, Guy; Liberator, Paul; Roemer, TerryChemistry & Biology (Cambridge, MA, United States) (2008), 15 (4), 363-374CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Natural products provide an unparalleled source of chem. scaffolds with diverse biol. activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chem. diversity, we survey natural products for antifungal, target-specific inhibitors by using a chem.-genetic approach adapted to the human fungal pathogen C. albicans and demonstrate that natural-product fermn. exts. can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochem. and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clin. relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action detn. of crude fermn. exts. by chem.-genetic profiling brings a powerful strategy to natural-product-based drug discovery.
- 11Xu, D., Ondeyka, J., Harris, G. H., Zink, D., Kahn, J. N., Wang, H., Bills, G., Platas, G., Wang, W., Szewczak, A. A., Liberator, P., Roemer, T., and Singh, S. B. (2011) Isolation, structure, and biological activities of fellutamides C and D from an undescribed Metulocladosporiella (Chaetothyriales) using the genome-wide Candida albicans fitness test J. Nat. Prod. 74, 1721– 1730 DOI: 10.1021/np200157311https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXovVegsbo%253D&md5=1092cdc7bc2967a84bafa70c05d2696eIsolation, structure, and biological activities of fellutamides C and D from an undescribed Metulocladosporiella (Chaetothyriales) using the genome-wide Candida albicans fitness testXu, Deming; Ondeyka, John; Harris, Guy H.; Zink, Deborah; Kahn, Jennifer Nielsen; Wang, Hao; Bills, Gerald; Platas, Gonzalo; Wang, Wenxian; Szewczak, Alexander A.; Liberator, Paul; Roemer, Terry; Singh, Sheo B.Journal of Natural Products (2011), 74 (8), 1721-1730CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society-American Society of Pharmacognosy)In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active exts. in the C. albicans fitness test (CaFT), which provides insight into the mechanism of action. A fermn. ext. of an undescribed species of Metulocladosporiella that inhibited proteasome activity in a C. albicans fitness test was identified. The chem. genomic profile of the ext. contained hypersensitivity of heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated from the ext. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 μg/mL and against fungal proteasome (IC50 0.2 μg/mL). Both compds. showed proteasome activity against human tumor cell lines, potently inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compd. treatment produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is crit. for the antifungal activity and that the 2 hydroxy groups are quant. important for potency.
- 12Rodriguez-Suarez, R., Xu, D., Veillette, K., Davison, J., Sillaots, S., Kauffman, S., Hu, W., Bowman, J., Martel, N., Trosok, S., Wang, H., Zhang, L., Huang, L. Y., Li, Y., Rahkhoodaee, F., Ransom, T., Gauvin, D., Douglas, C., Youngman, P., Becker, J., Jiang, B., and Roemer, T. (2007) Mechanism-of-action determination of GMP synthase inhibitors and target validation in Candida albicans and Aspergillus fumigatus Chem. Biol. 14, 1163– 1175 DOI: 10.1016/j.chembiol.2007.09.00912https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1amt77J&md5=92f53ba0b67720ee485d730fa129ac65Mechanism-of-Action Determination of GMP Synthase Inhibitors and Target Validation in Candida albicans and Aspergillus fumigatusRodriguez-Suarez, Roberto; Xu, Deming; Veillette, Karynn; Davison, John; Sillaots, Susan; Kauffman, Sarah; Hu, Wenqi; Bowman, Joel; Martel, Nick; Trosok, Steve; Wang, Hao; Zhang, Li; Huang, Li-Yin; Li, Yang; Rahkhoodaee, Fariba; Ransom, Tara; Gauvin, Daniel; Douglas, Cameron; Youngman, Phil; Becker, Jeff; Jiang, Bo; Roemer, TerryChemistry & Biology (Cambridge, MA, United States) (2007), 14 (10), 1163-1175CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)Mechanism-of-action (MOA) studies of bioactive compds. are fundamental to drug discovery. However, in vitro studies alone may not recapitulate a compd.'s MOA in whole cells. Here, we apply a chemogenomics approach in Candida albicans to evaluate compds. affecting purine metab. They include the IMP dehydrogenase inhibitors mycophenolic acid and mizoribine and the previously reported GMP synthase inhibitors acivicin and 6-diazo-5-oxo-L-norleucine (DON). We report important aspects of their whole-cell activity, including their primary target, off-target activity, and drug metab. Further, we describe ECC1385, an inhibitor of GMP synthase, and provide biochem. and genetic evidence supporting its MOA to be distinct from acivicin or DON. Importantly, GMP synthase activity is conditionally essential in C. albicans and Aspergillus fumigatus and is required for virulence of both pathogens, thus constituting an unexpected antifungal target.
- 13Xu, D., Sillaots, S., Davison, J., Hu, W., Jiang, B., Kauffman, S., Martel, N., Ocampo, P., Oh, C., Trosok, S., Veillette, K., Wang, H., Yang, M., Zhang, L., Becker, J., Martin, C. E., and Roemer, T. (2009) Chemical genetic profiling and characterization of small-molecule compounds that affect the biosynthesis of unsaturated fatty acids in Candida albicans J. Biol. Chem. 284, 19754– 19764 DOI: 10.1074/jbc.M109.019877There is no corresponding record for this reference.
- 14Richard, M. L. and Plaine, A. (2007) Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicans Eukaryot. Cell. 6, 119– 133 DOI: 10.1128/EC.00297-0614https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXitVersbc%253D&md5=afa6c59bf87ddae8c48facae2229da07Comprehensive analysis of glycosylphosphatidylinositol-anchored proteins in Candida albicansRichard, Mathias L.; Plaine, ArmelEukaryotic Cell (2007), 6 (2), 119-133CODEN: ECUEA2; ISSN:1535-9778. (American Society for Microbiology)A review that focuses on the function of glycosylphosphatidylinositol-anchored proteins in Candida albicans.
- 15Pittet, M. and Conzelmann, A. (2007) Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae Biochim. Biophys. Acta 1771, 405– 420 DOI: 10.1016/j.bbalip.2006.05.01515https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXjsVeru70%253D&md5=eb0ba642fce1f308d70f85d9458748fdBiosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiaePittet, Martine; Conzelmann, AndreasBiochimica et Biophysica Acta, Molecular and Cell Biology of Lipids (2007), 1771 (3), 405-420CODEN: BBMLFG; ISSN:1388-1981. (Elsevier Ltd.)A review. Like most other eukaryotes, Saccharomyces cerevisiae harbors a GPI anchoring machinery and uses it to attach proteins to membranes. While a few GPI proteins reside permanently at the plasma membrane, a majority of them gets further processed and is integrated into the cell wall by a covalent attachment to cell wall glucans. The GPI biosynthetic pathway is necessary for growth and survival of yeast cells. The GPI lipids are synthesized in the ER and added onto proteins by a pathway comprising 12 steps, carried out by 23 gene products, 19 of which are essential. Some of the estd. 60 GPI proteins predicted from the genome sequence serve enzymic functions required for the biosynthesis and the continuous shape adaptations of the cell wall, others seem to be structural elements of the cell wall and yet others mediate cell adhesion. Because of its genetic tractability S. cerevisiae is an attractive model organism not only for studying GPI biosynthesis in general, but equally for investigating the intracellular transport of GPI proteins and the peculiar role of GPI anchoring in the elaboration of fungal cell walls.
- 16Klis, F. M., Sosinska, G. J., de Groot, P. W., and Brul, S. (2009) Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence FEMS Yeast Res. 9, 1013– 1028 DOI: 10.1111/j.1567-1364.2009.00541.x16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlOis7bK&md5=03afa73bf2dfb2456c3420b9aa94c729Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulenceKlis, Frans M.; Sosinska, Grazyna J.; de Groot, Piet W. J.; Brul, StanleyFEMS Yeast Research (2009), 9 (7), 1013-1028CODEN: FYREAG; ISSN:1567-1356. (Wiley-Blackwell)A review. The cell wall of Candida albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, contg. c. 20 different protein species covalently linked to the skeletal layer. Most of them are GPI proteins. Coat proteins vary widely in function. Many of them are involved in the primary interactions between C. albicans and the host and mediate adhesive steps or invasion of host cells. Others are involved in biofilm formation and cell-cell aggregation. They further include iron acquisition proteins, superoxide dismutases, and yapsin-like aspartic proteases. In addn., several covalently linked carbohydrate-active enzymes are present, whose precise functions remain hitherto largely elusive. The expression levels of the genes that encode covalently linked cell wall proteins (CWPs) can vary enormously. They depend on the mode of growth and the combined inputs of several signaling pathways that sense environmental conditions. This is reflected in the unusually long intergenic regions of most of these genes. Finally, the precise location of several covalently linked CWPs is temporally and spatially regulated. It is concluded that covalently linked CWPs of C. albicans play a crucial role in fitness and virulence and that their expression is tightly controlled.
- 17Orlean, P. and Menon, A. K. (2007) Thematic review series: Lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids J. Lipid Res. 48, 993– 1011 DOI: 10.1194/jlr.R700002-JLR20017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXls1ejtLo%253D&md5=cdba29bd6397497683c83925fb816ca5GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipidsOrlean, Peter; Menon, Anant K.Journal of Lipid Research (2007), 48 (5), 993-1011CODEN: JLPRAW; ISSN:0022-2275. (American Society for Biochemistry and Molecular Biology, Inc.)A review. Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that were translocated across the ER membrane nd that display a GPI signal anchor sequence pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but 1 of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, the authors describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.
- 18Sütterlin, C., Horvath, A., Gerold, P., Schwarz, R. T., Wang, Y., Dreyfuss, M., and Riezman, H. (1997) Identification of a species-specific inhibitor of glycosylphosphatidylinositol synthesis EMBO J. 16, 6374– 6383 DOI: 10.1093/emboj/16.21.6374There is no corresponding record for this reference.
- 19Wang, Y., Dreyfuss, M., Ponelle, M., Oberer, L., and Riezman, H. A Glycosylphosphatidylinositol-anchoring inhibitor with an unusual tetracarboxocyclic sesterpene skeleton from the fungus Codinaea simplex. Tetrahedron 54, 6415– 6426. DOI: 10.1016/S0040-4020(98)00322-6There is no corresponding record for this reference.
- 20Hong, Y., Maeda, Y., Watanabe, R., Ohishi, K., Mishkind, M., Riezman, H., and Kinoshita, T. (1999) Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol J. Biol. Chem. 274, 35099– 35106 DOI: 10.1074/jbc.274.49.35099There is no corresponding record for this reference.
- 21Wiedman, J. M., Fabre, A. L., Taron, B. W., Taron, C. H., and Orlean, P. (2007) In vivo characterization of the GPI assembly defect in yeast mcd4–174 mutants and bypass of the Mcd4p-dependent step in mcd4Delta cells FEMS Yeast Res. 7, 78– 83 DOI: 10.1111/j.1567-1364.2006.00139.x21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhslyhuro%253D&md5=be87a6b02cd4021df9acfca6ebd728d4In vivo characterization of the GPI assembly defect in yeast mcd4-174 mutants and bypass of the Mcd4p-dependent step in mcd4Δ cellsWiedman, Jill M.; Fabre, Anne-Lise; Taron, Barbara W.; Taron, Christopher H.; Orlean, PeterFEMS Yeast Research (2007), 7 (1), 78-83CODEN: FYREAG; ISSN:1567-1356. (Blackwell Publishing Ltd.)Yeast mcd4-174 mutants are blocked in glycosylphosphatidylinositol (GPI) anchoring of protein, but the stage at which GPI biosynthesis is interrupted in vivo has not been identified, and Mcd4p has also been implicated in phosphatidylserine and ATP transport. The authors report that the major GPI that accumulates in mcd4-174 in vivo is Man2-GlcN-(acyl-Ins)PI, consistent with proposals that Mcd4p adds phosphoethanolamine to the 1st mannose of yeast GPI precursors. Mcd4p-dependent modification of GPIs can partially be bypassed in the mcd4-174/gpi11 double mutant and in mcd4Δ mutants by high-level expression of PIG-B and GPI10, which resp. encode the human and yeast mannosyltransferases that add the 3rd mannose of the GPI precursor. Rescue of mcd4Δ by GPI10 indicates that Mcd4p-dependent addn. of EthN-P to the 1st mannose of GPIs is not obligatory for transfer of the 3rd mannose by Gpi10p.
- 22Tsukahara, K., Hata, K., Nakamoto, K., Sagane, K., Watanabe, N. A., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly Mol. Microbiol. 48, 1029– 1042 DOI: 10.1046/j.1365-2958.2003.03481.x22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXktV2gsL4%253D&md5=1049da3e97c3df636219d903cc2f939cMedicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assemblyTsukahara, Kappei; Hata, Katsura; Nakamoto, Kazutaka; Sagane, Koji; Watanabe, Nao-aki; Kuromitsu, Junro; Kai, Junko; Tsuchiya, Mamiko; Ohba, Fuminori; Jigami, Yoshifumi; Yoshimatsu, Kentaro; Nagasu, TakeshiMolecular Microbiology (2003), 48 (4), 1029-1042CODEN: MOMIEE; ISSN:0950-382X. (Blackwell Publishing Ltd.)Glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small mol. inhibitors of the cell surface presentation of GPI-anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI-anchored cell wall mannoproteins. By means of a yeast cell-based screening procedure, we discovered a compd., 1-[4-butylbenzyl]isoquinoline (I), that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compd. resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage-dependent suppressor of the I-induced phenotypes. GWT1 knock-out cells showed similar phenotypes to I-treated wild-type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to I each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compd.
- 23Gaynor, E. C., Mondésert, G., Grimme, S. J., Reed, S. I., Orlean, P., and Emr, S. D. (1999) MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast Mol. Biol. Cell 10, 627– 648 DOI: 10.1091/mbc.10.3.62723https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXhvVyku7c%253D&md5=cc36193454987edf3eea47f0c292fa83MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeastGaynor, Erin C.; Mondesert, Guillaume; Grimme, Stephen J.; Reed, Steve I.; Orlean, Peter; Emr, Scott D.Molecular Biology of the Cell (1999), 10 (3), 627-648CODEN: MBCEEV; ISSN:1059-1524. (American Society for Cell Biology)Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a crit. role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temp.-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphol. defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. Mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.
- 24Dennehy, K. M. and Brown, G. D. (2007) The role of the beta-glucan receptor dectin-1 in control of fungal infection J. Leukoc. Biol. 82, 253– 258 DOI: 10.1189/jlb.120675324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXos12rtbk%253D&md5=369bd2e12247f8cc55c6a65bfaa63432The role of the β-glucan receptor Dectin-1 in control of fungal infectionDennehy, Kevin M.; Brown, Gordon D.Journal of Leukocyte Biology (2007), 82 (2), 253-258CODEN: JLBIE7; ISSN:0741-5400. (Federation of American Societies for Experimental Biology)A review. During fungal infection, a variety of receptors initiates immune responses, including TLR and the β-glucan receptor Dectin-1. TLR recognition of fungal ligands and subsequent signaling through the MyD88 pathway were thought to be the most important interactions required for the control of fungal infection. However, recent papers have challenged this view, highlighting the role of Dectin-1 in induction of cytokine responses and the respiratory burst. Two papers, using independently derived, Dectin-1-deficient mice, address the role of Dectin-1 in control of fungal infection. Saijo et al. (Nat. Immunol., 2007, 8, 39-46) argue that Dectin-1 plays a minor role in control of Pneumocystis carinii by direct killing and that TLR-mediated cytokine prodn. controls P. carinii and Candida albicans. By contrast, Taylor et al. (Nat. Immunol, 2007, 8, 31-38) argue that Dectin-1-mediated cytokine and chemokine prodn., leading to efficient recruitment of inflammatory cells, is required for control of fungal infection. Here, the authors argue that collaborative responses induced during infection may partially explain these apparently contradictory results. They propose that Dectin-1 is the first of many pattern recognition receptors that can mediate their own signaling, as well as synergize with TLR to initiate specific responses to infectious agents.
- 25Umemura, M., Okamoto, M., Nakayama, K., Sagane, K., Tsukahara, K., Hata, K., and Jigami, Y. (2003) GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast J. Biol. Chem. 278, 23639– 23647 DOI: 10.1074/jbc.M301044200There is no corresponding record for this reference.
- 26Watanabe, N. A., Miyazaki, M., Horii, T., Sagane, K., Tsukahara, K., and Hata, K. (2012) E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis Antimicrob. Agents Chemother. 56, 960– 971 DOI: 10.1128/AAC.00731-1126https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs1Gruro%253D&md5=a489c9d19296eaa8b65513de5b133ce3E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesisWatanabe, Nao-aki; Miyazaki, Mamiko; Horii, Takaaki; Sagane, Koji; Tsukahara, Kappei; Hata, KatsuraAntimicrobial Agents and Chemotherapy (2012), 56 (2), 960-971CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concns. (IC50s) of 0.3 to 0.6 μM but had no inhibitory activity against human Pig-Wp even at concns. as high as 100 μM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude ext. and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concns. above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.
- 27McLellan, C. A., Whitesell, L., King, O. D., Lancaster, A. K., Mazitschek, R., and Lindquist, S. (2012) Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity ACS Chem. Biol. 7, 1520– 1528 DOI: 10.1021/cb300235m27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XovFaktr0%253D&md5=f5d23f76cbc1942730dce3af6b2af115Inhibiting GPI Anchor Biosynthesis in Fungi Stresses the Endoplasmic Reticulum and Enhances ImmunogenicityMcLellan, Catherine A.; Whitesell, Luke; King, Oliver D.; Lancaster, Alex K.; Mazitschek, Ralph; Lindquist, SusanACS Chemical Biology (2012), 7 (9), 1520-1528CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biol. of GPI-anchor synthesis is difficult, esp. in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, the discovery of a new small mol. christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a crit. acyltransferase required for the biosynthesis of fungal GPI anchors, is reported. After delineating the target specificity of gepinacin using genetic and biochem. techniques, it was used to probe key, therapeutically relevant consequences of disrupting GPI anchor metab. in fungi. It was found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compd. results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture expts. to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concns. impaired filamentation and unmasked cell wall β-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepinacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi.
- 28Lee, M. C., Miller, E. A., Goldberg, J., Orci, L., and Schekman, R. (2004) Bi-directional protein transport between the ER and Golgi Annu. Rev. Cell Dev. Biol. 20, 87– 123 DOI: 10.1146/annurev.cellbio.20.010403.10530728https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVaqu7bM&md5=ae02d65edd306dafa84ef899b1d628cdBi-directional protein transport between the ER and GolgiLee, Marcus C. S.; Miller, Elizabeth A.; Goldberg, Jonathan; Orci, Lelio; Schekman, RandyAnnual Review of Cell and Developmental Biology (2004), 20 (), 87-123CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)A review. The endoplasmic reticulum (ER) and the Golgi app. comprise the 1st 2 steps in protein secretion. Vesicular carriers mediate a continuous flux of proteins and lipids between these compartments, reflecting the transport of newly synthesized proteins out of the ER and the retrieval of escaped ER residents and vesicle machinery. Anterograde and retrograde transport is mediated by distinct sets of cytosolic coat proteins, the COPII and COPI coats, resp., which act on the membrane to capture cargo proteins into nascent vesicles. Here, the authors review the mechanisms that govern coat recruitment to the membrane, cargo capture into a transport vesicle, and accurate delivery to the target organelle.
- 29Kodera, C., Yorimitsu, T., Nakano, A., and Sato, K. (2011) Sed4p stimulates Sar1p GTP hydrolysis and promotes limited coat disassembly Traffic 12, 591– 599 DOI: 10.1111/j.1600-0854.2011.01173.xThere is no corresponding record for this reference.
- 30Letourneur, F., Gaynor, E. C., Hennecke, S., Demolliere, C., Duden, R., Emr, S. D., Riezman, H., and Cosson, P. (1994) Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79, 1199– 1207 DOI: 10.1016/0092-8674(94)90011-6There is no corresponding record for this reference.
- 31Ma, W. and Goldberg, J. (2013) Rules for the recognition of dilysine retrieval motifs by coatomer EMBO J. 32, 926– 937 DOI: 10.1038/emboj.2013.41There is no corresponding record for this reference.
- 32Carvajal, E., van den Hazel, H. B., Cybularz-Kolaczkowska, A., Balzi, E., and Goffeau, A. (1997) Molecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genes Mol. Gen Genet. 256, 406– 415 DOI: 10.1007/s00438005058432https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhsl2juw%253D%253D&md5=fb7163231216cf3d46323b51f5490edeMolecular and phenotypic characterization of yeast PDR1 mutants that show hyperactive transcription of various ABC multidrug transporter genesCarvajal, E.; Van Den Hazel, H. B.; Cybularz-Kolaczkowska, A.; Balzi, E.; Goffeau, A.Molecular & General Genetics (1997), 256 (4), 406-415CODEN: MGGEAE; ISSN:0026-8925. (Springer-Verlag)Mutations at the yeast PDR1 transcriptional regulator locus are responsible for overexpression of the three ABC transporter genes PDR5, SNQ2 and YOR1, assocd. with the appearance of multiple drug resistance. The nucleotide sequences of 13 alleles of PDR1, comprising 6 multidrug resistance mutants, 1 intragenic suppressor and 6 wild types, have been detd. Single amino acid substitutions were shown to result from the mutations pdr1-2 (M308I), pdr1-3 (F815S), pdr1-6 (K302Q), pdr1-7 (P298A) and pdr1-8 (L1036W), whereas the intragenic suppressor mutant pdr1-100 is deleted for the two amino acids L537 and A538. An isogenic series of strains was constructed contg. the mutant alleles pdr1-3, pdr1-6 and pdr1-8 integrated into the genome. It was found that the levels of resistance to cycloheximide, oligomycin, 4-nitroquinoline-N-oxide and ketoconazole were increased in all three mutants. The increase was more pronounced in the pdr1-3 than in the pdr1-6 and pdr1-8 mutants. Studies of the activity of the promoters of the ABC genes PDR5, SNQ2 and YOR1 demonstrated that the combination of the PDR5 promoter and the pdr1-3 mutation resulted in the highest level of promoter induction. Concomitantly. the level of PDR5 mRNA, of Pdr5p protein, and of its assocd. nucleoside triphosphatase activity, was strongly increased in the plasma membranes of the PDR1 mutants. Again, the pdr1-3 allele was assocd. with a stronger effect than the pdr1-8 and pdr1-6 alleles. The locations of the mutations in the PDR1 gene indicate that at least three different regions distributed throughout the Pdr1p transcription factor may be mutated to generate a Pdr1p with considerably increased transcriptional activation potency. These gain-of-function mutations support the concept, recently proposed, that in members of the large family of yeast Zn2Cys6 transcription factors a central inhibitory domain exists (delineated by the pdr1-7, pdr1-6 and pdr1-2 mutations). This domain may interact in a locked conformation with a putative, more C-terminally located inhibitory domain (mutated in pdr1-3), and with the putative activation domain (mutated in pdr1-8).
- 33Sagane, K., Umemura, M., Ogawa-Mitsuhashi, K., Tsukahara, K., Yoko-o, T., and Jigami, Y. (2011) Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p J. Biol. Chem. 286, 14649– 14658 DOI: 10.1074/jbc.M110.193490There is no corresponding record for this reference.
- 34Zhu, Y., Vionnet, C., and Conzelmann, A. (2006) Ethanolaminephosphate side chain added to glycosylphosphatidylinositol (GPI) anchor by mcd4p is required for ceramide remodeling and forward transport of GPI proteins from endoplasmic reticulum to Golgi J. Biol. Chem. 281, 19830– 19839 DOI: 10.1074/jbc.M601425200There is no corresponding record for this reference.
- 35Miyazaki, M., Horii, T., Hata, K., Watanabe, N. A., Nakamoto, K., Tanaka, K., Shirotori, S., Murai, N., Inoue, S., Matsukura, M., Abe, S., Yoshimatsu, K., and Asada, M. (2011) In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds Antimicrob. Agents Chemother. 55, 4652– 4658 DOI: 10.1128/AAC.00291-1135https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2nsL%252FP&md5=cb380963b1f986ed2e0d72ed5067e19dIn vitro activity of E1210, a novel antifungal, against clinically important yeasts and moldsMiyazaki, Mamiko; Horii, Takaaki; Hata, Katsura; Watanabe, Nao-aki; Nakamoto, Kazutaka; Tanaka, Keigo; Shirotori, Syuji; Murai, Norio; Inoue, Satoshi; Matsukura, Masayuki; Abe, Shinya; Yoshimatsu, Kentaro; Asada, MakotoAntimicrobial Agents and Chemotherapy (2011), 55 (10), 4652-4658CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)E1210 is a new antifungal compd. with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clin. fungal isolates. E1210 showed potent activities against most Candida spp. (MIC90 of ≤0.008 to 0.06 μg/mL), except for Candida krusei (MICs of 2 to >32 μg/mL). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC90 of 0.13 μg/mL). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 μg/mL), Scedosporium prolificans (MIC of 0.03 μg/mL), and Paecilomyces lilacinus (MICs of 0.06 μg/mL) among the compds. tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been obsd. with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.
- 36Poulain, D. and Jouault, T. (2004) Candida albicans cell wall glycans, host receptors and responses: elements for a decisive crosstalk Curr. Opin. Microbiol. 7, 342– 349 DOI: 10.1016/j.mib.2004.06.01136https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmt1Omsbg%253D&md5=f563b4eb9e95bb8616d8e58dc707d8c1Candida albicans cell wall glycans, host receptors and responses: elements for a decisive crosstalkPoulain, Daniel; Jouault, ThierryCurrent Opinion in Microbiology (2004), 7 (4), 342-349CODEN: COMIF7; ISSN:1369-5274. (Elsevier Ltd.)A review. Candida albicans has adapted to live on the mucosal surfaces of animals. The human species has accepted it. By contrast to numerous other commensals, C. albicans has a prominent ability to invade virtually all tissues of a host presenting with natural or acquired defects in homeostasis. C. albicans uses considerable energy to synthesize glycans, which are present either as polymers or as glyconjugates. These glycan mols. play a prominent role in the biol. of C. albicans by controlling the structure and plasticity of the cell wall, and are also involved in yeast-host interactions. These glycans are recognized as non-self by host innate and adaptative immunity. The signal they induce in the host depends on the glycan code, which is detd. by the nature of the sugar, the anomer type of linkage and branching, and the length of the oligosaccharide chains. However, this model is not static because the nature of the C. albicans mol. carrying such glycan codes and their expression at the cell wall surface also dets. the host response, and, in turn, the regulation of cell wall glycan arrangement dynamics in C. albicans depends on host stimuli. Candida glycans therefore play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and resistance and infection. A goal of current research concerning the virulence attributes of C. albicans will be to det. to what extent this species is able to regulate its glycan code as a response to the host.
- 37Wheeler, R. T. and Fink, G. R. (2006) A drug-sensitive genetic network masks fungi from the immune system PLoS Pathog. 2, e35 DOI: 10.1371/journal.ppat.0020035There is no corresponding record for this reference.
- 38Ishibashi, K., Yoshida, M., Nakabayashi, I., Shinohara, H., Miura, N. N., Adachi, Y., and Ohno, N. (2005) Role of anti-beta-glucan antibody in host defense against fungi FEMS Immunol. 44, 99– 109 DOI: 10.1016/j.femsim.2004.12.01238https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVajsr8%253D&md5=9ad0c2384b1b3f5833b60d8b88d9f592Role of anti-β-glucan antibody in host defense against fungiIshibashi, Ken-Ichi; Yoshida, Masaharu; Nakabayashi, Iwao; Shinohara, Hiroyasu; Miura, Noriko N.; Adachi, Yoshiyuki; Ohno, NaohitoFEMS Immunology and Medical Microbiology (2005), 44 (1), 99-109CODEN: FIMIEV; ISSN:0928-8244. (Elsevier B.V.)The authors have recently detected an anti-β-glucan antibody in normal human and normal mouse sera. The anti-β-glucan antibody showed reactivity to pathogenic fungal Aspergillus and Candida cell wall glucan. Anti-β-glucan antibody could bind whole Candida cells. It also enhanced the candidacidal activity of macrophages in vitro. The anti-β-glucan antibody titer of DBA/2 mice i.v. administered either Candida or Aspergillus solubilized cell wall β-glucan decreased remarkably dependent on dose. Moreover, in deep mycosis patients, the anti-β-glucan antibody titer decreased, and this change correlated with clin. symptoms and other parameters such as C-reactive protein. It was suggested that the anti-β-glucan antibody formed an antigen-antibody complex and participated in the immune response as a mol. recognizing pathogenic fungi.
- 39Kushida, H., Nakajima, S., Uchiyama, S., Nagashima, M., Kojiri, K., Kawamura, K., and Suda, H. (1999) Antifungal substances BE-49385 and process for their production. U.S. Patent 5,928,910.There is no corresponding record for this reference.
- 40Hirano, A., Sugiyama, E., Kondo, H., Suda, H., Ogawa, H., and Kojiri, K. (2001) Sesterterpene derivatives exhibiting antifungal activities U.S. Patent 6,303,797 B1.There is no corresponding record for this reference.
- 41Sütterlin, C., Escribano, M. V., Gerold, P., Maeda, Y., Mazon, M. J., Kinoshita, T., Schwarz, R. T., and Riezman, H. (1998) Saccharomyces cerevisiae GPI10, the functional homologue of human PIG-B, is required for glycosylphosphatidylinositol-anchor synthesis Biochem. J. 332, 153– 159There is no corresponding record for this reference.
- 42McConville, M. J. and ad Ferguson, M. A. J. (1993) The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes Biochem. J. 294, 305– 32442https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlvF2hs70%253D&md5=23f9cf6089c12012eaa42693daafc790The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotesMcConville, Malcolm J.; Ferguson, Michael A. J.Biochemical Journal (1993), 294 (2), 305-24CODEN: BIJOAK; ISSN:0264-6021.A review with >200 refs. on the structure, function and biosynthesis of glycosyl-phosphatidylinositol (GPI) anchors in the title organisms. There may be significant differences in the function of GPI protein anchors in unicellular vs. metazoan organisms. Some parasitic protozoa synthesize exotic GPI-related structures which are not attached to proteins. Evolutionary aspects of the GPI family are also discussed.
- 43Hata, K., Horii, T., Miyazaki, M., Watanabe, N. A., Okubo, M., Sonoda, J., Nakamoto, K., Tanaka, K., Shirotori, S., Murai, N., Inoue, S., Matsukura, M., Abe, S., Yoshimatsu, K., and Asada, M. (2011) Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis Antimicrob. Agents Chemother. 55, 4543– 4551 DOI: 10.1128/AAC.00366-1143https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtl2ns7fE&md5=ff015a73a4108a15fe6474a7411a6044Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosisHata, Katsura; Horii, Takaaki; Miyazaki, Mamiko; Watanabe, Nao-aki; Okubo, Miyuki; Sonoda, Jiro; Nakamoto, Kazutaka; Tanaka, Keigo; Shirotori, Syuji; Murai, Norio; Inoue, Satoshi; Matsukura, Masayuki; Abe, Shinya; Yoshimatsu, Kentaro; Asada, MakotoAntimicrobial Agents and Chemotherapy (2011), 55 (10), 4543-4551CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)E1210 is a 1st-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and ref. antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater redn. in the no. of oral CFU than the control treatment. In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice. E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice. E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to det. E1210's potential for the treatment of disseminated fungal infections are indicated.
- 44Lee, A. Y., St. Onge, R. P., Proctor, M. J., Wallace, I. M., Nile, A. H., Spagnuolo, P. A., Jitkova, Y., Gronda, M., Wu, Y., Kim, M. K., Cheung-Ong, K., Torres, N. P., Spear, E. D., Han, M. K., Schlecht, U., Suresh, S., Duby, G., Heisler, L. E., Surendra, A., Fung, E., Urbanus, M. L., Gebbia, M., Lissina, E., Miranda, M., Chiang, J. H., Aparicio, A. M., Zeghouf, M., Davis, R. W., Cherfils, J., Boutry, M., Kaiser, C. A., Cummins, C. L., Trimble, W. S., Brown, G. W., Schimmer, A. D., Bankaitis, V. A., Nislow, C., Bader, G. D., and Giaever, G. (2014) Mapping the cellular response to small molecules using chemogenomic fitness signatures Science 344, 208– 211 DOI: 10.1126/science.125021744https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXlslSmtbw%253D&md5=543c8d40d54c94f789521ed29859d403Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness SignaturesLee, Anna Y.; St. Onge, Robert P.; Proctor, Michael J.; Wallace, Iain M.; Nile, Aaron H.; Spagnuolo, Paul A.; Jitkova, Yulia; Gronda, Marcela; Wu, Yan; Kim, Moshe K.; Cheung-Ong, Kahlin; Torres, Nikko P.; Spear, Eric D.; Han, Mitchell K. L.; Schlecht, Ulrich; Suresh, Sundari; Duby, Geoffrey; Heisler, Lawrence E.; Surendra, Anuradha; Fung, Eula; Urbanus, Malene L.; Gebbia, Marinella; Lissina, Elena; Miranda, Molly; Chiang, Jennifer H.; Aparicio, Ana Maria; Zeghouf, Mahel; Davis, Ronald W.; Cherfils, Jacqueline; Boutry, Marc; Kaiser, Chris A.; Cummins, Carolyn L.; Trimble, William S.; Brown, Grant W.; Schimmer, Aaron D.; Bankaitis, Vytas A.; Nislow, Corey; Bader, Gary D.; Giaever, GuriScience (Washington, DC, United States) (2014), 344 (6180), 208-211CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small mols. affects biol. and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compd. in vivo to profile 3250 small mols. in a systematic and unbiased manner. We identified 317 compds. that specifically perturb the function of 121 genes and characterized the mechanism of specific compds. Global anal. revealed that the cellular response to small mols. is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chems., and biol. processes.
Supporting Information
Supporting Information
ARTICLE SECTIONSThe following file is available free of charge on the ACS Publications website at DOI: 10.1021/id5000212.
Tables S1–S4 contain general strain information, mammalian cytotoxicity results, and expanded bacterial spectrum testing; Figures S1–S7 contain additional data describing CaFT analysis, heterozygote spot testing, Aspergillus zones of inhibition, time–kill assays, conditional mutant response, and Mcd4 inhibitor stability in plasma (PDF)
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