The first potent inhibitor of squalene synthase: a profound contribution of an ether oxygen to inhibitor-enzyme interaction

Biller, Scott A.; Sofia, Michael J.; DeLange, Barbara; Forster, Cornelia; Gordon, Eric M.; Harrity, Thomas; Rich, Lois C.; Ciosek, Carl P.

doi:10.1021/ja00022a050

Cited by 72 publications

(48 citation statements)

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“…It is proposed that PSPP or a derivative (X'), or PPi, or a combination of these metabolites signal the process(es) which modulate the rate of HMG-CoA reductase (HMGR) irreversible inactivation. SQ 32709 and L-694,599 are squalene synthase inhibitors (11,12).…”

Section: Resultsmentioning

confidence: 99%

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

Girón

Havel

Watson

1994

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

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The regulation of mevalonic acid synthesis requires both nonsterol isopentenoid and sterol regulatory signal molecules. A primary target of this multivalent control process is the enzyme which catalyzes mevalonate synthesis:3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reduc- It has been proposed that mammalian mevalonic acid (MVA) synthesis is controlled negatively by the availability of sterols and nonsterol isopentenoids (1) (Fig. 1) The cells were used at %90o confluence. Specific manipulations and/or growth conditions are described in the figure and table legends. a-Toxin Isolation. a-Toxin was isolated and purified (18) from S. aureus (provided by S. Harshman, Vanderbilt University, Nashville, TN). A typical purification yielded 20-30 mg of a-toxin per liter of culture, with a specific activity of =5 x 107 hemolytic units/mg of a-toxin.Cell Permeabilization. CHO-K1 cells were plated in 25-cm2 flasks and incubated for 2 days in McCoy's 5A medium with 5% fetal bovine serum. On the day ofthe experiment the cells were washed twice with phosphate-buffered saline (without Ca2+ and Mg2+) containing 2.5% (wt/vol) fatty acid-depleted bovine serum albumin and were perforated by incubation for 15 min at 370C with a-toxin (5 t&g/ml) in permeabilization buffer (20 mM potassium Mops, pH 7.0/250 mM mannitol/1 mM potassium ATP/3 mM MgCl2/5 mM potassium glutathione). Afterwards, the cells were washed twice with ice-cold incubation buffer (5 mM KH2PO4, pH 7.0/51 mM KCI/10 mM MgCl2/1 mM potassium ATP/1404 mM mannitol/10 mM potassium succinate/5 mM potassium glutamate/5 mM potassium glutathione). Then 1 ml of 370C incubation buffer (supplemented with 0.5 mM NADP, 5 mM potassium glucose-6-phosphate, 11 units of glucose-6-phosphate dehydrogenase, 0.5 mM NAD, 10 mM creatine Abbreviations: FPP, farnesyl 1-diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl 1-diphosphate; MVA, mevalonic acid; WSIP, water-soluble isopentenoid phosphate ester. 6398The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Section: Resultsmentioning

confidence: 99%

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

Girón

Havel

Watson

1994

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

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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%

“…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. Several phosphinylformate analogs of FPP showed micromolar activity in blocking cholesterol synthesis in freshly isolated rat hepatocytes and in human skin fibroblasts (28).…”

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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“…The design of the FPP-based inhibitors of FT is similar to that which was exploited in the design of potent squalcnc synthase inhibitors [Biller et al, 1991[Biller et al, , 1992Ciosek et al, 19931. Compounds I and I1 (Fig.…”

Section: Inhibitor Designmentioning

confidence: 99%

Ras farnesylation as a target for novel antitumor agents: Potent and selective farnesyl diphosphate analog inhibitors of farnesyltransferase

Manne

Ricca

Brown

et al. 1995

Drug Development Research

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Protein prenylation is increasingly recognized as an important mechanism by which functional association of proteins t o membranes is mediated. Ras proteins, regulators of cell proliferation and differentiation, are among the proteins that undergo farnesylation, one of the two prenylation modifications known. Since ras proteins are activated into hyperactive oncogenic versions in a wide variety of human cancers, agents that d o w n modulate ras activity could be antineoplastic. Therefore, inhibitors of farnesyltransferase have the potential t o be of therapeutic value as anticancer agents due t o their ability t o block ras processing and hence its function. W e describe the identification of two farnesyl pyrophosphate (FPP) analogs that are potent and selective inhibitors of farnesyltransferase. While showing n o toxicity t o untransformed cells, a pivaloyloxyrnethyl ester of one of these inhibitors blocked ras mediated transformation of NIH 3T3 cells. In addition, both the ester and its parent acid inhibited ras farnesylation as measured by incorporation of labeled mevalonate into ras proteins in whole cells. Thus, this i s the first report of an FPP analog t o show biological activity by inhibiting ras processing i n whole cells. (0 1995 Wile"-Liss, Inc.

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The first potent inhibitor of squalene synthase: a profound contribution of an ether oxygen to inhibitor-enzyme interaction

Cited by 72 publications

References 0 publications

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

Mevalonate-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase function in alpha-toxin-perforated cells.

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

Ras farnesylation as a target for novel antitumor agents: Potent and selective farnesyl diphosphate analog inhibitors of farnesyltransferase

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