Inhibition of 2,3-oxidosqualene-lanosterol cyclase in Candida albicans by pyridinium ion-based i

A NTIMICROBIAL A GENTS AND C HEMOTHERAPY,Apr.1996,p.1044–1047Vol.40,No.4 0066-4804/96/$04.00ϩ0Copyright᭧1996,American Society for MicrobiologyInhibition of2,3-Oxidosqualene-Lanosterol Cyclase in Candidaalbicans by Pyridinium Ion-Based InhibitorsROBERT C.GOLDMAN,1*DOROTHY ZAKULA,1JOHN O.CAPOBIANCO,1BRADLEY A.SHARPE,2AND JOHN H.GRIFFIN2Anti-infective Research Division,Abbott Laboratories,Abbott Park,Illinois60064-3500,1and Department ofChemistry,Stanford University,Stanford,California94305-50802Received10October1995/Returned for modification7November1995/Accepted12January1996 The N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1-ylpyridinium and N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1-ylpicolinium cations were evaluated for their ability to inhibit2,3-oxidosqualene-lanosterolcyclase activity in Candida albicans.Both compounds inhibited fungal growth,were fungicidal,and resulted inthe accumulation of squalene epoxide concurrent with a decrease in ergosterol,monomethyl sterols,andlanosterol,as was expected for the specific inhibition of2,3-oxidosqualene-lanosterol cyclase activity.Thesecompounds are electron-poor aromatic mimics of a monocyclized transition state or high-energy intermediateformed from oxidosqualene,which may explain their selective action.The ergosterol biosynthetic pathway is an important target of antifungal drugs.The azole antifungal agents,such as keto-conazole,itraconazole,andfluconazole,inhibit lanosterol14␣-demethylase.The imidazole or triazole moiety of these inhib-itors ligates the ferric ion of this heme-containing enzyme, while the hydrophobic portions of the inhibitors interact with the substrate binding site(25,26).In contrast,the allylamine antifungals(naftifine and terbinafine)inhibit squalene epoxi-dase via a lipophilic interaction with the enzyme,resulting in the accumulation of squalene(22,23).2,3-Oxidosqualene-lanosterol cyclase(EC5.4.99.7)catalyzes the complex cyclization-rearrangement of oxidosqualene to lanosterol and serves as another potential target site for anti-fungal drug inhibition(7,11,17,18).Several classes of fungal cyclase inhibitors have been reported(4–6,8,12–14,27,28), one of which includes the mechanism-based inactivator29-methylidene-2,3-oxidosqualene,which covalently modifies ver-tebrate cyclase(1–3).The gene(ERG7)from Candida albicans (9)and Saccharomyces cerevisiae(15,24)was cloned(19)and sequenced.The sequences of these and other members of the triterpene cyclase family are rich in tryptophan and tyrosine residues which bear electron-rich aromatic side chains.There-fore,compounds with high affinity for electron-rich aromatic groups,such as electron-poor aromatics,may be cyclase inhib-itors.The fungal enzyme may differ from mammalian and plant enzymes,since suicide substrate[3H]29-methylidene-2,3-ox-idosqualene will label mammalian and plant enzymes but not the enzyme from S.cerevisiae(1).In this report,we describe the antifungal activity of cyclase inhibitors,the inhibition of 2,3-oxidosqualene-lanosterol cyclase in C.albicans cells,and the subsequent antifungal effects of these novel pyridinium ion-based inhibitors.Antifungal activity of inhibitors.The N-(4E,8E)-5,9,13-tri-methyl-4,8,12-tetradecatrien-1-ylpyridinium(compound1)and N-(4E,8E)-5,9,13-trimethyl-4,8,12-tetradecatrien-1-ylpicolinium (compound2)cations(Fig.1)were chosen for study as elec-tron-poor aromatic mimics of a monocyclized transition state or high-energy intermediate formed from oxidosqualene.Am-photericin B was used as a control antifungal compound.An-tifungal activity was tested at37ЊC in yeast nitrogen base (YNB;Difco)containing0.5%glucose and buffered with0.01 M phosphate buffer(final pH,7.0).Cells were inoculated into microtiter wells(1,000CFU per well)containing twofold serial dilutions of drugs,and the MIC was determined after48h of incubation(Table1).Both compounds1and2were active against a panel of test organisms and approached the MIC of amphotericin B.They were also active against the one ampho-tericin B-resistant isolate in the panel.The MIC of compound 1ranged from0.78␮g/ml(1.5␮M)to6.25␮g/ml(12.6␮M) and that of compound2ranged from1.56␮g/ml(3.2␮M)to 12.5␮g/ml(26␮M).These values are significantly lower than those recently reported for the activity of22,23-epoxy-2-aza-2,3-dihydrosqualene(5)and slightly higher than those re-ported for benzophenone analogs(18),both of which inhibit the cyclization of oxidosqualene.Although the method is not yet formally approved,antifungal activity was also examined in RPMI medium(21)by a method proposed by the National Committee for Clinical Laboratory Standards(Table1).Both compounds gave MICs in RPMI equal to those in YNB or MICs that were at most twofold different from those in YNB. Except for two cases showing a fourfold difference,amphoter-icin B values were identical in both media.Antifungal activity againstfluconazole-sensitive and-resistant C.albicans(Table 2)obtained from M.A.Pfaller(Department of Pathology, University of Iowa)was also tested in RPMI medium.Al-though slight differences were observed,no definitive evidence indicating cross-resistance was obtained.At5and10times their MICs,both compounds were fungicidal for C.albicans CCH442(Fig.2).Inhibition of2,3-oxidosqualene-lanosterol cyclase in C.al-bicans cells.Inhibition of2,3-oxidosqualene-lanosterol cyclase was observed when C.albicans CCH442was treated with either compound1or2.Cells were grown in YNB(Difco)plus glucose,washed,and resuspended to an A420of5.0in25mM Na-K phosphate buffer,pH6.5,containing1%glucose.The analysis of sterol synthesis proceeded as described previously (10),with a10-min preincubation with the drug occurring prior to the addition of2␮Ci of[U-14C]acetate(0.5mM[0.8mCi/ mmol]final concentration)for2h at30ЊC.Cells washed by centrifugation were saponified at90ЊC for2h,and non-sapon-ifiable lipid(NSL)was extracted into petroleum ether.An*Corresponding author.Mailing address:Anti-infective Research Division,Abbott Laboratories,100Abbott Park Rd.,Abbott Park,IL 60064-3500.Phone:(708)937-4477.Fax:(708)938-6603.Electronic mail address:robert.goldman@.1044internal standard,5␣-dihydro[4-14C]testosterone (2.9mCi/mmol),was added to each tube prior to the extraction of NSL.The petroleum ether was evaporated to dryness under a stream of N 2,and the residue taken up in cyclohexane and spotted on 20-by-20-cm silica gel plates (with preconcentration zones).The plates were developed in chloroform or chloroform with 3%(vol/vol)ethanol,and spots were located and quantitated with a radioactive thin-layer chromatography scanner (Radi-omatic).Radioactive spots comigrating with desmethyl sterols (ergosterol),4-monomethyl sterols,4,4-dimethyl sterols (lanos-terol),oxidosqualene,and squalene were normalized to control values,with the internal standard counts being used as thereference.Values were determined in triplicate,and standard deviations were determined.The level of oxidosqualene increased concurrently with a decrease in the levels of ergosterol,lanosterol,and 4-mono-methyl sterols when the cells were treated with increasing doses of compound 1(Fig.3A)or 2(Fig.3B).Oxidosqualene accounted for less than 5%of the total NSL in control cells but amounted to 70or 88%of the NSL when cells were treated with 6␮g of compound 1or 2per ml,respectively.The level ofFIG.1.Conversion of 2,3-oxidosqualene to lanosterol and the structures of pyridinium ion-based inhibitor compounds 1and 2.FIG.2.Fungicidal activity of compounds 1and 2.C.albicans CCH442grow-ing exponentially in YNB medium at 37ЊC was treated with 5or 10times the MIC of compound 1(A)or 2(B),and the number of CFU per milliliter was deter-mined in triplicate by plating the cells on yeast extract-peptone-dextrose me-dium.Standard deviations are given,although the error bars are too small to be represented in some cases.Open bars,control;crosshatched bars,5ϫMIC;horizontally hatched bars,10ϫMIC.TABLE 1.Antifungal activity of squalene cyclase inhibitorcompounds 1and 2StrainMIC (␮g/ml)aCompound 1Compound 2Amphotericin BCandida albicans ATCC 1023112.5(6.25) 6.25(3.12) 1.56(1.56)Candida albicans 579a12.5(6.25) 6.25(3.12) 1.56(1.56)Candida albicans CCH44212.5(12.5) 6.25(6.25) 1.56(1.56)Candida albicans ATCC 38247 3.12(3.12) 1.56(3.12)50(50)Candida albicans ATCC 6237612.5(6.25) 6.25(3.12) 1.56(1.56)Candida tropicalis NRRL-Y-112 6.25(3.12) 3.12(1.56) 1.56(1.56)Candida kefyr ATCC 28838 6.25(3.12) 1.56(1.56) 3.12(0.78)Torulopsis glabrata ATCC 155453.12(1.56)0.78(0.78) 3.12(0.78)Cryptococcus albidus ATCC 3414012.5(6.25) 6.25(6.25) 1.56(1.56)Saccharomycescerevisiae GSI-36 3.12(3.12) 1.56(1.56) 1.56(1.56)Aspergillus niger ATCC164041.56(6.25)3.12(3.12)1.56(1.56)aMICs were determined by microbroth dilution with YNB medium.Values in parentheses were determined with RPMI medium.TABLE 2.Antifungal activity of squalene cyclase inhibitor compounds 1and 2against fluconazole-sensitive and -resistantC.albicansStrainMIC (␮g/ml)aCompound 1Compound 2FluconazoleC.albicans OY2-76420.5C.albicans OY2-79421C.albicans OY3-551684C.albicans OY8-4716832C.albicans 1378-171684C.albicans 1378-481688C.albicans 04-812264C.albicans ATCC 900281620.5aThe MIC was determined by microtiter dilution with RPMI medium.V OL .40,1996NOTES 1045total NSLs also increased with increasing dose and then de-clined as the dose of either compound was increased further.An unknown drug-induced metabolite migrating just ahead of lanosterol accounted for less than 18%of the label incorpo-rated in the presence of either drug and was best separated from lanosterol by including ethanol to 3%(vol/vol)during thin-layer chromatography of the NSLs.This unknown metab-olite migrated with an R f value similar to that of 2,3,22,23-dioxidosqualene,a metabolite which appears to accumulate when oxidosqualene cyclase activity is below normal (19).No evidence for significant direct or feedback inhibition of squalene epoxidase or inhibition of lanosterol demethylation,as occurred with azasqualene analogs,was observed (5).The level of detected squalene is low in the whole cell system (3.5%Ϯ0.6%of total NSL)and ranged between 3and 5%of the NSLs in drug-treated cells.The 50%inhibitory concentrations of compound 1were 1.6␮g/ml,or 3.2␮M (r 2ϭ0.99),and 2.1␮g/ml,or 4.2␮M (r 2ϭ0.99),with the inhibition of ergosterol or ergosterol plus lanosterol formation,respectively,being used,compared with 5.7␮g/ml,or 11.8␮M (r 2ϭ0.95),and 4.4␮g/ml,or 9.1␮M (r 2ϭ0.96),for compound 2(Fig.4).The 50%inhibitory concentrations corresponded reasonably well with the relative MICs for C.albicans CCH442(12.5␮g/ml [25.1␮M]and 6.25[12.9␮M]for compounds 1and 2,respec-tively).No ergosterol,monomethyl sterols,or lanosterol was detected when cells were treated with 25to 50␮g of either compound per ml.Oxidosqualene represented 80to 85%of the NSLs under these conditions,with the remainder ac-counted for by the unknown,which is likely to be 2,3,22,23-dioxidosqualene on the basis of its R f value (19).The two pyridinium ion-based compounds inhibited the er-gosterol pathway in C.albicans at the oxidosqualene cycliza-tion step.Although total NSL values did show a transient increase,the fraction of NSL recovered as oxidosqualene in-creased throughout the dose range,concurrent with a decrease in the level of ergosterol and lanosterol,the two major post-cyclization products.Both compounds were fungicidal,per-haps because of the accumulation of squalene epoxide con-taining the reactive epoxide functionality.Neither compound caused leakage of previously accumulated radiolabeled ami-noisobutyric acid (16)from cells when the compounds were tested at 25␮g/ml,indicating that direct damage to membrane integrity was not part of their mode 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