Isoprenoid (phosphinylmethyl)phosphonates as inhibitors of squalene synthetase

Biller, Scott A.; Forster, Cornelia; Gordon, Eric M.; Harrity, Thomas; Scott, William; Ciosek, Carl P.

doi:10.1021/jm00118a001

Cited by 88 publications

(24 citation statements)

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“…Modestly active inhibitors of squalene synthase have been described previously, and most are analogs of FPP (25)(26)(27)(28)(29) or are ammonium analogs of proposed carbocationic intermediates in the conversion of presqualene diphosphate to squalene (30,31). One potent exception to this was a famesyl phosphinylphosphonate ether analog (29), which was a competitive inhibitor of rat liver squalene synthase with a K1 of 37 nM.…”

Section: Discussionmentioning

confidence: 99%

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Bergstrom

Kurtz

Rew

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

315

158

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Three closely related fungal metabolites, The mammalian isoprenoid pathway not only produces sterols but also produces dolichol, ubiquinone, the farnesyl group of heme A, the farnesyl and geranylgeranyl groups of prenylated proteins, and the isopentenyl side chain of isopentenyl adenine. The pathways for the synthesis of these other isoprenoids diverge from the synthesis ofcholesterol either at or before the farnesyl diphosphate (FPP) branch point. Thus, squalene synthase, which catalyzes the reductive dimerization of 2 mol of FPP to 1 mol of squalene (2, 3), is the first committed step in sterol synthesis. A specific inhibitor of squalene synthase should serve to inhibit cholesterol synthesis and not adversely affect the synthesis of other isoprenoids. FPP, the substrate for squalene synthase, is water soluble and may be readily metabolized (4). Thus, squalene synthase offers a potential target for the safe and specific inhibition of cholesterol synthesis.In this report we describe the isolation, structure, physical characterization, and biological properties of three structurally similar fungal metabolites that are potent inhibitors of squalene synthase. These metabolites, zaragozic acid A (5-7), zaragozic acid B (8, 9), and zaragozic acid C (10-12), had been reported previously only in the patent literature; however, during the review process of this manuscript, three manuscripts (13-15) appeared on the squalestatins: squalestatin I is identical with zaragozic acid A, squalestatin II is des-4'-acetylzaragozic acid A, and squalestatin III is des-6-acylzaragozic acid A. This class of squalene synthase inhibitors has potential utility as cholesterol-lowering agents. MATERIALS AND METHODSZaragozic Acid A, Cultures, and Media. An unidentified sterile fungal culture, ATCC 20986, isolated from a water sample taken from the Jalon river in Zaragoza, Spain (hence the name zaragozic acids), was used to produce zaragozic acid A. The culture was maintained at 25°C on medium B agar slants composed of 4 g of yeast extract, 10 g of malt extract, 4 g of dextrose, and 20 g of agar per liter at pH 7.0.Zaragozic acid A was produced in a two-tiered fermentation process consisting of mycelial growth and development in medium A of ref. 1 and product formation in medium C. Medium C contained 5 g of malt extract, 1 g of peptone, 15 g of dextrose, 1 g of KH2PO4, and 0.5 g of MgSO4 7H20 per liter. Fermentations consisted of mycelial growth in medium A for 72 hr at 250C with agitation, followed by inoculation (5-10%) of medium C. Maximum product was obtained from 14-day agitated fermentations at 250C.Isolation of Zaragozic Acid A. To isolate zaragozic acid A, 23 liters of harvested broth was filtered through Celite, and the mycelial cake was extracted twice with 7 liters of 50%o aqueous methanol. The filtrate was combined with the extracts, diluted with water to a final composition of 25% methanol, and adsorbed on a 1.5-liter column of Mitsubishi HP-20 resin. After a column wash with 6 liters of4:6 (vol/vol) methanol/water, crud...

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Section: Discussionmentioning

confidence: 99%

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Bergstrom

Kurtz

Rew

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

315

158

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“…Crude microsomal membranes from human cells and quickfrozen fungal cells were prepared as previously described (8,22). Squalene synthetase activity in these samples was measured by using a radiochemical assay (1).…”

Section: Materlals and Methodsmentioning

confidence: 99%

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

et al. 1993

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Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme.

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“…Pellets were resuspended on ice in 0.2 ml of breakage buffer using a glass homogenizer and were stored frozen at -70'C. Squalene synthetase activity in microsomes was assayed at 30'C under N2 using a gas chromatographic assay (19). RESULTS Cloning, Complementation Analysis, and Subcloning.…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning and characterization of the yeast gene for squalene synthetase.

Jennings

Tsay

Fisch

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

137

108

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Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway. The structural gene for this squalene by a two-step reductive condensation of two molecules of FDP. It has been isolated from many sources and its reaction mechanism has been investigated in some detail (3, 4). Squalene synthetase solubilized from membranes of bakers' yeast, Saccharomyces cerevisiae, has been described (4-8) and the purified enzyme was reported to have a molecular mass of "50 kDa (5-8). Yeast with defects in squalene synthetase were identified by screening a collection of mutants blocked in ergosterol biosynthesis (9). Mutants designated erg9 were unable to convert radiolabeled mevalonate into squalene but did accumulate farnesol, a breakdown product of FDP. Because these squalene synthetase mutants were also obligate ergosterol auxotrophs, they were used in a phenotype complementation approach to clone the ERG9 gene. In this report, we describe the structure of squalene synthetase deduced from its primary sequence and explore the consequences of genomic deletion of this gene in S. cerevisiae. * MATERIALS AND METHODSStrains. The following strains of S. cerevisiae were used in this study: DC67 (MA Ta ERG9 leu24 adel lys2 cir,) was from J. Hicks; W303-1A (MA Ta) and W303-1B (MATa) (both ERG9 leu2-3,112 ade2-1 ura3-1 trpl-J his3-11,15) were from R. Rothstein; SGY336 (MATa erg9-1) was from F. Karst; JRY527 (MATa ERG9 ura3-52 ade2-101 his3-d200 lys2-801 Met-) was from J. Rine; SC14089 (MATa ERG9) was from J.Tkacz; SGY1161 an ERG9/ERG9 diploid was from a mating of W303-1A and W303-1B; SGY969 an ERG9/erg9 diploid was constructed from SGY336 by mating to W303-1B; SGY1011 (MATa erg9 leu2-3,112 ade2-1 ura3-1 his3-11,15) was a segregant from SGY969. Standard Plasmid-borne disruptions of the ERG9 gene were constructed by replacement of a 1.0-kb EcoRI/Sca I fragment of

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Isoprenoid (phosphinylmethyl)phosphonates as inhibitors of squalene synthetase

Abstract: In this paper, we describe our initial findings on the preparation and biological evaluation of isoprenoid (phosphinylmethyl)phosphonates (PMPs), stable analogues of farnesyl diphosphate (FPP, 1), as inhibitors of squalene synthetase. Squalene synthetase123"4 catalyzes the

Cited by 88 publications

References 0 publications

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

Molecular cloning and characterization of the yeast gene for squalene synthetase.

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