inhibitors of ergosterol biosynthesis and growth of the ... · vol. 265, no. 9. issue of march 25,...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 9. Issue of March 25, pp. 4989-4996,199O Printed in U.S.A.

Inhibitors of Ergosterol Biosynthesis and Growth of the Trypanosomatid Protozoan Crithidia fascicuZuta*

(Received for publication, June 12, 1989)

Mohammad D. Rahman and Robert A. Pascal, Jr.* From the Department of Chemistry, Princeton University, Princeton, New Jersey 08544

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 pM for 24-thia-5a,20t- cholestan-38-01 (2), 0.009 FM for 24-methyl-24-aza- Sa,2O.+holestan-38-01 (3), 0.95 MM for (20,21),(24,- 25)~bis-(methylene)-5a-cholestan-3&ol (4), 0.13 pM for 22-aza-Sa,20&cholestan-3fi-o1 (5), and 0.3 pM for 23-azacholestan-3-01 (7). 23-Thia-5a-cholestan-38-01 (6) had no effect on protozoan growth at concentrations as high as 20 PM.

Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-38-01, ergosta-7,24(28)-dien-3/l-01, ergosta- 5,7,22,24(28)-tetraen-38-01, cholesta-8,24-dien-3@- 01, and, in an unusual finding, 14m-methyl-cholesta- 8,24-dien-38-01 were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-38-01, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C- 24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost- 7-en-3/3-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3&ol and ergosta-7,24- (28)-dien-38-01, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the Az4(“) side chain double bond. When C. famiculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.

Ergosterol (1) is the principal sterol of the parasitic trypanosomatid flagellates (l-5), and it differs from cholesterol, the predominant mammalian sterol, by the presence of a 24- methyl group and A7 and AZ2 double bonds. The three enzy-

* This work was supported by National Institutes of Health Grant AI24146 and bv an Alfred P. Sloan Research Fellowshiu (to R. A. P.). The costs of publication of this article were defrayed ;n part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

matic reactions which introduce the extra methyl group and the AZ2 double bond of ergosterol have no counterpart in mammalian sterol biosynthesis (6), and they may be regarded as potential targets for new antiparasitic drugs. The three reactions are S-adenosylmethionine-dependent side chain alkylation to give a 24-methylene group, reduction of the A24(28) double bond to give a methyl group, and desaturation at C- 22,23 to give the AZ2 double bond. Specific inhibitors of these enzymes should have no effect on a mammalian host, but such compounds would either halt protozoan sterol biosynthesis completely or at the very least force the organisms to function with unusual sterols. An entirely analogous strategy is well established in the area of fungicide development, where many inhibitors of ergosterol biosynthesis have been shown to be potent antifungal agents (7).

\ 22 CH,

SCHEME 1

Among the most interesting compounds are heteroatom- substituted sterols, particularly sterols with nitrogen substi- tution in the side chain, and the mechanism of action of these inhibitors has been closely examined. A variety of 23-, 24-, and 25-azasterols have been shown to inhibit the S-adenosylmethionine:sterol C-24 methyltransferase (henceforth re- ferred to as the A24-methyltransferase) of yeast (8, 9), and similar results have been obtained in higher plants (10, 11). All such azasterols are positively charged at physiological pH, and they are thought to be analogs of carbonium ion inter- mediates in the methyl transfer reactions. The finding that sterols bearing side chain sulfonium and arsonium groups also inhibit the A24-methyltransferase provides additional support for this concept (11). Some azasterols have also been reported to inhibit the A24(2s) reduction (8), but no specific inhibitors of the C-22,23 desaturation have been found, although general inhibitors of the cytochromes P-450 have been reported to interfere with this process (12, 13).

In order to assess the value of inhibitors of ergosterol biosynthesis as antiprotozoal agents, we have begun to study the effects of such compounds on growth and sterol biosynthesis in the representative trypanosomatid Crithidia fusciculutu. We report herein (a) a detailed analysis of the normal sterol composition of C. fusciculutu, including the isolation and characterization of ergosterol and five minor sterols from these organisms, (b) the synthesis of a variety of cholestanols containing nitrogen, sulfur, or cyclopropanes in the sterol side chain, (c) the inhibition of growth of C. fusciculutu by these compounds at nanomolar to micromolar concentrations in the

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Inhibitors of Ergosterol Biosynthesis and Growth of C. fasciculata

culture medium, and (d) the effects of these compounds on the sterol composition of the protozoa, including evidence that certain of the compounds are specific inhibitors of C-24 alkylation and Az4@) reduction.

MATERIALS AND METHODS

General

Melting points and UV-visible absorption spectra were obtained as previously described (14). ‘H NMR spectra were recorded on chloroform-d solutions of sterols on JEOL GSX-500 and GSX-270 NMR spectrometers operating at 500 MHz or 270 MHz, respectively. Low and high resolution direct inlet mass spectra were obtained on a Kratos MS50 mass spectrometer. Ergosterol (1) and 20-(hydroxymethyl)pregna-1,4-diene-3-one were obtained from Aldrich. The sterol inhibitors employed in this study (compounds 2-7) are illustrated in Fig. 1. 3/3-Tetrahydropyranyloxy-5a-pregnan-20-one, 24- thia-5o(,20&cholestan-3P-o1(2), and 24-methyl-24-aza-5a,2O&cholestan-30-01 (3) were synthesized as described previously (15). The syntheses of (20,21),(24,25)-bis(methylene)-501-cholestan-3~-ol (4) (procedures a-d), 22-aza-5a-20[-cholestan-3@-ol (5) (procedure e), 23-thia-5a-cholestan-30-01 (6) (procedures f-i), 23-azacholestan-3-01 (7) (procedure j), and ergost-7-en-3p-ol (procedure k) are described below. The syntheses of compounds 4-7 are outlined in Figs. 2 and 3. Cholesta-8,24-dien-3P-ol (zymosterol) was isolated from yeast as described previously (16).

(a)3P-Tetrahydropyranyloxy-21-nor-5~-cholest-24-en-2O-one(S)- n-Butyllithium (1.08 ml of a 2.5 M solution in hexane, 2.7 mmol) was added to a solution of diisopropylamine (0.35 ml, 2.5 mmol) in dry

.o&sT Ho&y k 6 HH 7

FIG. 1. Inhibitors of ergosterol biosynthesis.

Ho&AHo& A A FIG. 2. Synthesis of (20,21),(24,25)-bis(methylene)-5a-

cbolestan-38-01 (4) and 22-aza-5a,20&cholestan-3&ol (5). The letters on the arrows refer to procedures described in the text.

THF’ (10 ml) under argon at -78 “C. After 15 min, 3P-tetrahydropyranyloxy-5oc-pregnan-20-one (1.00 g, 2.5 mmol) in THF (3 ml) was added, and after 30 min this was followed by 4-bromo-2-methyl-2- butene (0.35 ml, 2.7 mmol). The solution was allowed to warm to room temperature overnight, methanol (20 ml) was added, and the mixture was poured into ether (150 ml). The ethereal solution was washed with water and 1 N NaHC03, and it was dried over MgS04. Evaporation of the solvent left a yellow oil which was chromatographed on silica gel (100:7 hexane/ethyl acetate). Concentration of the fractions exhibiting RF = 0.17 on silica gel TLC (2O:l hexane/ ethyl acetate) gave compound 8 (0.80 g, 68% yield), mp 104-105 “C. ‘H NMR d 5.07 (m, lH, 24-H), 4.71 (m, lH, THP methine), 3.92 and 3.50 (m’s, 2H, THP OCH,), 3.61 (m, lH, 3a-H), 1.68 and 1.60 (s’s, 6H, 26-H3 and 27-Ha), 0.80 (s, 3H, 19-Hs), 0.57 (s, 3H, 18-Ha); MS, m/z 470 (M+, 17), 455 (M-CHs, 6), 386 (M-DHP, 4), 369 (48), 287 (lo), 257 (24), 111 (61), 85 (100). Exact mass 470.3758, calculated for C31H5003 470.3760. Also isolated was the double alkylation product 3P-tetrahydropyranyloxy-17-(3-methyl-2-butenyl)-21-nor-5a-cholest-24-en-20-one (TLC RF = 0.24,0.20 g, 15% yield), mp 120-121 “C. ‘H NMR 6 5.07 (m, 2H vinyl H’s), 4.71 (m, lH, THP methine), 3.92 and 3.50 (m’s, 2H, THP OCH2), 3.61 (m, lH, 3a-H), 1.67, 1.63, 1.58, 1.54 (s’s, 12H, methyls of the two side chains), 0.79 (s, 3H, 19-Ha), 0.56 (s, 3H, 18-H& Exact mass (M-C5H, ion) 469.3672, calculated for C3iH4a03 469.3672.

@) 3P-Tetrahydropyranyloxy-5a-cholesta-20(21),24-diene (9). n- Butyllithium (0.52 ml of a 2.5 M solution in hexane, 1.30 mmol) was added to a solution of methyltriphenylphosphonium bromide (0.46 g, 1.29 mmol) in benzene (10 ml), and the mixture was refluxed for 1 h. Compound 8 (0.20 g, 0.43 mmol) was added, and reflux was continued for 20 h. After an extractive workup similar to that above, a red oil was obtained. This material was chromatographed on silica gel (2O:l hexane/ethyl acetate). The appropriate fractions were combined and concentrated to dryness, and the residue was recrystallized from methanol to give compound 9’ (0.12 g, 60% yield), mp 78-80 “C. ‘H NMR d 5.06 (m, lH, 24-H), 4.86 and 4.77 (s’s, 2H, 21-HZ), 4.65 (m, lH, THP methine), 3.92 and 3.50 (m’s, 2H, THP OCHZ), 3.61 (m, lH, 3o1-H), 1.68 and 1.60 (s’s, 6H, 26-Ha and 27-Ha), 0.80 (s, 3H, 19- H3), 0.54 (s, 3H, 18-Hs); MS, m/z 468 (M’, l), 384 (M-DHP, 8), 369 (21), 273 (76), 257 (29), 161 (22), 93 (100). Exact mass 468.3954, calculated for C32H5202 468.3957.

(c) 5a-Cholesta-20(21),24-dien-3@-ol (IO)-Compound 9 (56 mg,

i The abbreviations used are: THF, tetrahydrofuran; TLC, thin layer chromatography; GC, gas chromatography; MS, mass spectrometry; TMS, trimethylsilyl; RRT, relative retention times.

*A referee expressed concern that epimerization at C-17 might have occurred during the Wittig olefination of compound 8. A single crystal x-ray structure of the product 9 was obtained and it is illustrated as Structure 1. The configuration at C-17 is normal (17a- H). In the two remaining steps in the synthesis of compound 4 (hydrolysis of the tetrahydropyranyl ether and Simmons-Smith cyclopropane synthesis) there is no possibility of epimerization at C-17 (or at any other position in the steroid nucleus), so the stereochemistry of 4 is also established by this x-ray structure determination. A single crystal of 9 measuring 0.05 X 0.15 X 0.52 mm was used for x- ray measurements. Crystal data: C35H5202; orthorhombic, space qoup P2i2,2,; a = 6.750 (2) A, b = 11.367 (4) A, and c = 37.977 (13) A, V = 2913.8 (1.4) A3, 2 = 4, &cd = 1.07 g/cm3. Intensity measurements were made with 3” 5 20 % 114” by using graphite monochromated Cu Ka radiation (X = 1.54178 A) at room temperature on a Nicolet R3m diffractometer. A total of 2315 unique reflections were measured, of which 1493 were considered to be observed [lFd 1 3u(F,)]. The structure was solved by direct methods using the SHELXTL software. Refinement converged at R = 0.095, R, = 0.109. We thank Dr. Donna Van Engen for the structure determination. Full details will be published elsewhere.

STRUCTURE 1


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Inhibitors of Ergosterol Biosynthesis and Growth of C. fasciculata 4991

0.12 mmol) was refluxed in 10 ml of methanol containing three drops of concentrated hydrochloric acid for 3 h. After an extractive workup similar to that above, the organic phase was concentrated to dryness, and the residue was recrystallized from methanol to give compound 10 (30 ma, 65% yield), mp 93-94 “C (melts), 105 “C (clears). ‘H NMR d 5.10 (m:lH, 24-H), 4.86 and 4.77 (s’s, 2H, 21-H*), 3.58 (m, lH, 3a- H). 1.67 and 1.64 (s’s. 6H. 26-HX and 27-H,). 0.80 (s. 3H. 19-HI). 0.54 (s;3H, 18H3); MS, m/z 384 (I+, 7), 369 (M-CH, il), 273 (5ij, 255 (23), 109 (57), 93 (100). Exact mass 384.3381, calculated for C&H,,0 384.3384.

(d) (20,21),(24,25)-Bis(methylenej-5a-cholestan-3/3-ol(4)-A crystal of iodine was added to Zn-Cu couple (69 mg) in ether (20 ml), and the mixture was stirred until the brown color disappeared. Compound 10 (25 mg, 0.07 mmol) and methylene iodide (279 mg) were added, and the mixture was refluxed for 5 days. After an extractive workup similar to above, the organic phase was concentrated and subjected to preparative TLC (silica gel). The appropriate band was scraped off the plate and eluted with chloroform. The extract was concentrated to dryness, and the residue was recrystallized from methanol to give compound 4 (8 mg, 30% yield), mp 91-93 “C (melts), 103 “C (clears). ‘H NMR (assignments are based on a homoscalar correlated two- dimensional ‘H NMR (COSY) spectrum of 4) 6 3.59 (m, lH, 3~u-H), 1.02 and 1.00 (s’s, 6H, 26-H3 and 27-H& 0.81 (6, 3H, 19-Hz), 0.70 (s, 3H, 18-Hs), 0.63 (m, 2H, 21-H and 21’-H, trans to the steroidnucleus), 0.36 (m. 1H. 24-H). 0.32 (m. 1H. 28-H. truns to C-23). 0.18 and -0.09 (m’s,‘ZH, 21-H and 21’-H, cis to the steroid nucleus), -0.18 (m, lH, 28-H, cis to C-23); MS, m/z 412 (M+, 3%), 397 (M-CH,, 6), 273 (loo), 255 (28), 161 (49), 147 (38), 135 (36), 121 (48), 107 (73), 105 (28), 95 (70), 94 (38), 93 (58). Exact mass 412.3710, calculated for C&Ha0 412.3709. This material showed a single component by silica gel TLC (1:l benzene/ether, RF = 0.53).

fe) 22-Az&a,2O~-cholestan-3/3-ol (5)--3&Tetrahydropyranyloxy- 5a-pregnan-20-one (0.402 g, 1 mmol) was dissolved in a mixture of methanol and THF (2:1,30 ml); isoamyl amine (0.087 g, 1 mmol) and NaCNBH3 (100 mg) were added, and the mixture was stirred at room temperature for 48 h. The reaction mixture was diluted with water and extracted with ether. The ether layer was acidified with 10% HCl to precipitate 3P-tetrahydropyranyloxy-22-azacholestanol hydrochlo- ride and the resulting salt was washed with ether. Dilute NaOH was added to the precipitate, and the mixture was extracted with ether. The ether layer was concentrated to dryness, and the residue (0.24 g) was dissolved in ethanol (20 ml) and concentrated HCl (1 ml) and refluxed for 3 h. The reaction mixture was diluted with water and extracted with ether. The ether layer was washed with solutions of NaHC03 and water and dried over Na2S04. Concentration gave a crude solid (0.2 g) which was chromatographed on a silica gel column (lo:1 hexane-ethyl acetate), and the appropriate fractions were combined to give compound 5 (150 mg, 38%) as a solid which was recrystallized from methanol; mp 128-129 “C (melts), 135 “C (clears). ‘H NMR 6 3.58 (m, lH, 3cu-H), 2.69 (m, lH, 20-H), 2.42 (m, 2H, 23- Hz), 1.06 (d, J = 6 Hz, 21-Hz), 0.89 and 0.88 (d’s, 6H, J = 6 Hz, 26- H, and 27-H3), 0.79 (s, 3H, 19-Ha), 0.66 (s, 3H, 18-Hz); MS, m/z 389 (M+, l%), 388 (M-H, 3), 374 (M-CH3, 32), 332 (9), 140 (8), 114

FIG. 3. Synthesis of 23-thia-5a-cholestan-3@-ol(6) and 23- azacholestan-3-ol(7). The letters on the arrows refer to procedures described in the text.

(Me,CHCH2CH2NH=CHCH3, 100). Exact mass 388.3575, calculated for C26H,sN0 (M-H) 388.3576.

(f) 20-(HydroxymethyU-5t-pregnan-3-one (11)-A solution of 20- (hydroxymethyl)-pregna-1,4-diene-3-one (1.0 g, 3.05 mmol) in ethanol (40 ml) was hydrogenated (40 psi) over 10% Pd on C (lo%, 200 mg) in a Parr apparatus overnight. The reaction mixture was filtered through celite and concentrated to dryness to give white solid which was recrystallized from methanol (0.98 g, 96%), mp 122-123 “C (melts), 128 “C (clears). ‘H NMR 6 3.64 (dd, 1 H, J = 11, 3 Hz, 22- H), 3.37 (dd, lH, J = 11, 7 Hz, 22-H), 1.05 (d, 3H, J = 7 Hz, 21-Hz), 1.02 (s, 3H, 19-H,), 0.70 (s, 3H, 18-H3); MS, m/z, 332 (M+, 9%), 317 (M-CH, 3), 299 (M-CHB-OH, 3), 231(12), 203 (lo), 108 (14), 85 (28), 83 (42), 78 (100). Exact mass 332.2710, calculated for C&HX.O~ 332.2715.

(g) 20-(p-Toluenesulfonyloxymethyl)-5(-pregnan-3-one (12)--Com- pound 11 (0.62 g, 1.87 mmol) was dissolved in dry pyridine (25 ml), the solution was cooled in an ice-salt bath, and tosyl chloride (0.715 g, 3.73 mmol) was added with stirring. After 20 h at -10 “C the reaction was poured into ice water (150 ml). A white sticky precipitate formed immediately which was collected by filtration. This material was taken un in ether (the tosic acid remained undissolved). Concen- tration of the ether gave the product 12 (0.6 g, 66%) which was recrvstallized from methanol: mn 179-80 “C (discolored). ‘H NMR 6 7.78”and 7.34 (AA’BB’ svstem, 4H, Ar-HI), 3:96 (dd, 1H; J = 9,3 Hz, 22-H), 3.78 (dd, lH, J =9, 6 Hz, 22-H), 2.44 (s, 3H, Ar-CH3), 1.00 (s, 3H. 19-H,). 0.98 (d. 3H. J = 6 Hz. 21-Ha). 0.64 (s. 3H. 18-Hs). MS, m/i 486 $I+, 2&j, 314 (M-tosic’acid, 58), 299 (25), 281 (39), 231 (64), 213 (45j, 161 (54), 121 (53), 107 (65), 91 (go), 83 (100). Exact mass 486.2797. calculated for C&H,,O,S 486.2798.

(h) 23-This-5&cholestan-3-&e- (13)-Sodium isobutylthiolate (0.56 g, 5 mmol) was added to a solution of compound 12 (0.25 g, 0.51 mmol) in argon saturated ethanok the solution was stirred for 30 min and then refluxed for 3 h. The reaction mixture was poured into ether (100 ml), and the ethereal solution was washed with 1 N NaOH containina a small amount of sodium dithionite. The organic layer was dried over Na&O., and concentrated to give a yellow residue which was chromatographed on a silica gel column (chloroform). The appropriate fractions were combined and the solvent was evaporated to yield compound 13 (120 mg, 60%). ‘H NMR b 2.72-2.61 (m, 2H, 22-Hz), 2.36 (d, 2H, J = 7 Hz, 24-H*), 1.06 (d, 3H, J = 6 Hz, 21-Ha), 1.01 (5&isomer) and 0.99 (5a-isomer) (s’s, 3H, 19-H& 0.98 (d, 6H, J = 7 Hz, 26-H3 and 27-H3), 0.69 (5@-isomer) and 0.68 (5o-isomer) (s’s, 3H, 18-Ha); MS, m/z 404 (M+, 7%), 314 (M-Me$ZXKX-LS, 4), 315 (M- Me$HCH,SH, 4), 272 (100). Exact mass 404.3090, calculated for C,GH,,OS 404.3096.

(i) 23-This-5a-ch&stan-3R-ol (6) and 23-Thia-54-chalestan-3c- , 01-A solution of compound 13 (70 mg, 0.17 mmol) and sodium borohydride (50 mg) in ethanol (LO ml) was stirred for 1 h. Water (5 ml) and then ether (100 ml) were added, and the mixture was washed with saturated NaHC03 and water, dried over Na2S04, and concentrated to give compound 6 and its 5-epimer which were recrystallized from ether (60 mg, 85%); mp 112-114 “C (melts), 120 “C (clears). ‘H NMR d 3.62 (m, lH, 3-H), 2.62 (dd, lH, J = 12, 3 Hz, 22-H), 2.36 (dd, 2H, J = 7, 2 Hz, 23-H& 2.22 (dd, lH, J = 12, 9 Hz, 22-H), 1.05 (d. 3H. J = 7 Hz, 21-H3), 0.97 (d, 6H, J = 7 Hz, 26-H3 and 27-Hz), 6.91 (3&5P-isomer) and 0:79 (3/3,5a-isomer) (s’s, 3H, 19-Hz), 0.65 and 0.64 (s’s, 3H, 18-H& MS, m/z 406 (M+, 5%), 317 (M-Me&HCH&, 3), 316 (M-MelCHCHZSH, 4), 274 (M-sidechain-H, 100). Exact mass 406.3262, calculated for &H,,OS, 406.3264. This mixture of isomers was senarated bv orenarative TLC (Analtech Uniplate Taper Plate, 1:l benzene/etherj, w’lth RF 0.60 and 0.70, respectively, for the 3&5a- and 3ol,5@-isomers. 3@-5cu-isomer (6): ‘H NMR 6 3.62 (m, lH, 3a-H), 2.62 (dd, lH, J = 12, 3 Hz, 22-H), 2.36 (dd, 2H, J = 7, 2 Hz, 23-I-U 2.24 (dd, lH, J = 12, 9 Hz, 22-H), 1.05 (d, 3H, J = 6 Hz, 21-W, 0.97 (d. 6H. J = 7 Hz, 26-Hs and 27-Hs), 0.79 (s, 3H, 19-Ha), 0.65 (s, 3H, i8-H3): 3a,5@isomer: ‘H NMR 6 3.62 (m, lH, 30-H), 2.63 (dd, 1H J = 12, 3 Hz, 22-H), 2.35 9dd, 2H, J = 7, 2 Hz, 23-H*), 2.22 (dd, 1H, J = 12,9 Hz, 22-H), 1.05 (d, 3H, J = 6 Hz, 21-H& 0.98 (d, 6H, J = 7 Hz. 26-H* and 27-H,). 0.91 (s. 3H. 19-Hs), 0.64 (s, 3H. 18-H& The stereochemical assignment of the 3&501- and So,BP-isomers is based on the conmparison of the angular methyl chemical shifts with those of closely related compounds of known stereochemistry, and on angular methyl chemical shift calculations which employed the table of Zurcher (36).

cj) 23-Azacholestan-3-01 (7) (Mixture of 3/3,5a- and 3q5@Iso- mers)-Isobutylamine (73 mg, 1 mmol) was added to a stirred solution of compound 12 (160 mg, 0.33 mmol) in ethanol (25 ml) under argon. The solution was heated with stirring at 70 “C for 2 days and then


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4992 Inhibitors of Ergosterol Biosynthesis and Growth of C. fasciculata

refluxed overnight. The solvent was evaporated, and the residue was uurified bv nrenarative TLC (Analtech Unialate Taner Plate). The . <.* appropriate band was eluted to give 23-azacholestan:3-one as an oil (55 mg). Without further purification, this material was reduced with NaBHe, as described for the preparation of compound 6, to yield 35 ma (27%) of comnound ‘7, which was recrystallized from methanol; mp i33-135 “C (melts), 142 “C (clears). ‘H NMR 6 3.62 (m, lH, 3- H), 2.61 (m, lH, 22-H), 2.41 (m, 2H, 23-H and NH), 2.33 (m, lH, 23- H), 2.24 (dd, lH, J = 12, 9 Hz, 22-H), 0.96 (d, 3H, J = 6 Hz, 21-Ha), 0.91 (3a,5P-isomer) and 0.79 (30,5a-isomer) (s’s, 3H, 19-Hs), 0.89 (d, 6H, J = 6 Hz, 26-Hn and 27-Hs), 0.65 and 0.64 (s’s, 3H, 18-Hz); MS, m/i 389 (M+,‘l%), 374 (M-CH;,. l), 346 (M-Me&H, 2), 149 (lo), 86 (CH,NHCH,CHMe,. 100). Exact mass 389.3649. calculated for i&&NO 389.3657.-’

(k) Ergost-7-en-3/3-ol-Ergosterol(l.0 g) was dissolved in benzene/ ethanol (l:l, 40 ml) and hydrogenated at room temperature and 45 psi HZ pressure overnight using freshly prepared Raney Ni. The mixture was filtered over celite, the solvent was evaporated, and the product was recrystallized from methanol, mp 144-145 ‘C [lit. (17) 145-146 “Cl. ‘H NMR 6 5.15 (m, lH, 7-H), 3.59 (m, lH, 3a-H), 0.92 (d, 3H, J = 7 Hz, 21-H.& 0.85 (d, 3H, J = 7 Hz, 28-Ha), 0.79 (s, 3H, 19.HZ), 0.78 and 0.77 (d’s, 6H J = 6.8 Hz, 26-H3 and 27-Ha), 0.52 (s, 3H, 18-Ha); MS, m/z 400 (M’, lOO%), 285 (M-CH,, 26), 273 (15), 255 (51), 107 (26), 55 (25).

Culture of C. fa.sciculata

The growth medium and general culture conditions for C. foscicu- lata (ATCC 12857) were as described previously (18). Sterols were added to the culture medium as solutions in dimethyl sulfoxide; equal amounts of dimethyl sulfoxide were added to all cultures in a given series of experiments. Flasks containing 50 ml of the growth medium, and the desired amount of sterol inhibitors or other additives were inoculated with aliquots of C. fasciculatu (usually 1 X 10’ organisms). The cultures were incubated with shaking at 25 “C, and growth was monitored by changes in absorbance at 535 nm, with an absorbance change of 1.0 corresponding to a cell density of 7 X 10’ organisms/ ml.

Zsolation and Analysis of Sterol Mixtures

For the isolation of sterols from C. jasciculata, cells were harvested by centrifugation, and the cell paste was saponified by treatment with refluxing 15% ethanolic KOH (at least 4 ml KOH solution/g of cells) for 3 h. The reaction mixture was diluted with water, and extracted three times with hexane. The combined hexane extracts were washed with brine and dried over NazS04. Evaporation of the solvent gave the crude nonsaponifiable lipids. This material was subjected to gas chromatographic (GC) analysis, or, alternatively, purified further as described below before analysis.

The monohydroxysterols were obtained from the nonsaponifiable lipids by preparative thin layer chromatography on plates of Silica Gel GF (Uniplate Taper Plates, Analtech, Newark, DE; solvent: 25:l CHClJmethanol). The broad sterol band(s) was visualized bv snrav- ing the TLC plates with rhodamine 6G (0.1% in acetone) and illu- minating with ultraviolet light. The sterol band(s) was scraped off the plate, and the silica1 gel was extracted with ether and methanol. Concentration of the extract gave a sterol mixture free of most other lipid components.

Prior to GC or GC-MS analysis the sterols were converted to the corresponding trimethylsilyl (TMS) ether derivatives by treatment with N,O-bis(trimethylsilyl)acetamide and pyridine. Ordinary GC analyses of sterol TMS ethers were carried out with capillary columns (BP1 stationary phase, 25 m x 0.53 mm (Scientific Glass Engineering, Austin, TX); Supelcowax 10 stationary phase, 15 m X 0.53 mm (Supelco, Bellefont, PA)) in a Hewlett-Packard 5890A chromatograph with a flame ionization detector. Isothermal conditions were em- ploved; typical temneratures were 250 “C for the BP1 column and 246 “C for-the Supelcowax 10 column. GC-MS analyses were carried out on either a Kratos MS50 mass spectrometer or a Hewlett-Packard 5970 system (BP1 capillary, 12.5 m x 0.32 mm).

RESULTS

The Sterols of C. fasciculata-Although ergosterol has long been known to be the major sterol of C. fa.sciculata (l-3), a more detailed analysis of the sterol composition of these protozoa has not been carried out. The total sterols were isolated from a l-liter culture of C. fusciculatu, and this

mixture was analyzed by GC and GC-MS of its TMS ether derivative. The sterol TMS ethers were poorly resolved on the nonpolar BP1 capillary column, but Supelcowax 10 (260 “C) gave excellent separation. Six well-resolved sterol peaks, designated A-F were observed in the chromatogram (see Table I). Components B-D cochromatographed with authentic samples of the TMS ethers of cholesta8,24dien- 3fi-01, ergosterol, and ergost-7-en-3@-01, and the mass spectra of these components were consistent with these assignments. Each of these components was subsequently isolated and characterized as the free sterol by ‘H NMR and mass spectrometry (see below). Components A, E, and F did not co- chromatograph with any available standards, but all three were isolated and characterized as the free sterols by ‘H NMR and low and high resolution mass spectrometry (see below). On the basis of the spectroscopic data, components A, E, and F were identified as 14a-methyl-5a-cholesta-8,24-dien-3P-o1, ergosta-7,24(28)-dien-3/3-01, and ergosta-5,7,22,24(28)-tetraen-3/3-01, respectively. As expected, ergosterol (1, sterol C) was the most abundant sterol found in untreated C. fasciculutu; it comprised 7580% of the total sterols. The other components were each present at levels of 2-6% of the total sterols, but the precise values for each of these components were somewhat variable from experiment to experiment.

Inhibition of Growth of C. fusciculutu by Cholestunol Deriv- atives 2-7-Duplicate flasks of growth medium containing a variety of concentrations of the chosen inhibitor (usually four concentrations, plus a control with none) were inoculated with equal numbers of C. fusciculutu, and these cultures were incubated at 25 “C. Growth was monitored spectrophotomet- rically at intervals of 6-10 h until the control incubation achieved a LL& > 1.0, which was near the end of the expo- nential phase. After preliminary experiments to determine the approximate concentration for 50% inhibition of growth (&), the experiments were repeated twice using concentrations of inhibitor which encompassed the estimated IbO, so that this parameter could be determined more accurately. Compounds 2-5 and 7 were potent inhibitors of growth at submicromolar concentrations in the growth medium. The I50 values were 0.32 pM for 2, 0.008 pM for 3, 0.95 pM for 4, 0.13 FM for 5, and 0.3 pM for 7. Compound 7 is a mixture of 3j$5a- and 3cY,5P-isomers; it is not known if one or both are active. Compound 6 had no effect on protozoan growth at concentrations as high as 20 FM.

Preliminary Analysis of Sterol Composition of C. fasciculutu Grown in the Presence of Compounds 2-7-Gas chromatographic analyses of the sterols from inhibitor-treated cultures of C. fusciculutu revealed dramatically altered sterol distributions. Tables II and III summarize the analyses of sterols

TABLE I Gas chromatographic relative retention times observed for sterols

isolated from untreated C. faxiculata

GC peak RRT of the TMS &he@ Structural assignment for free stem1

ii

E F

1.26 1.36 1.45 1.56

1.70 1.81

14a-Methyl-5a-cholesta-8,24-dien-3/3-01’ 5o-Cholesta-8,24-dien-3@-ol (zymosterol)d Ergosterol” Ergost-7-en-3@-old Ergosta-7,24(28)-dien-3/3-01’ Ergosta-5,7,22,24(28)-tetraen-3P-ol

a Supelcowax 10 capillary column; 260 “C. * 3/3-Trimethylsilyloxy-5acholestane = 1.00. ‘Assignment based on the ‘H NMR and mass spectra of the

isolated material. d Assignment based on comparison of the chromatographic prop-

erties, ‘H NMR spectrum, and mass spectrum of the isolated material with those of an authentic sample.


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TABLE II Effects of compounds 2-4 on the growth of C. fasciculata and on the sterol content of the nonsaponifiable lipids

Inhibitof NOIF2 Compound 3

0.009 f 0.002

Inhibitor concentration (PM) 0 0.01 0.05 Wet weight of cells (g) 4.2 1.3 0.3 Mass of isolated sterols (mg) (ap- 6.0 2.0 0.5

proximate) Distribution of sterols (%)

Ergosterol (sterol C) 75.2 10.0 1.6 14a-Methylcholesta-8,24-dien- 5.2 1.7 3.2

3/3-01 (A) Cholesta-8,24-dien-3fl-ol (B) 6.1 2.7 Cl.0 Ergost-7-en-BP-01 (D) 4.5 11.6 <LO Ergosta-7,24(28)-dien-3/3-ol (E) 2.7 Cl.0 Cl.0 Ergo&a-5,7,22,24(28)-tetraen- 6.7 8.9 Cl.0

3p-01 (F) Cholesta-5,7,24-trien-SO-01 (G) <l.O 50.0 88.3 Other <LO 15.0 6.9

’ Cont. (PM) to give 50% growth inhibition (I&.

Compound 2 Compound 4

0.32 f 0.05 0.95 2 0.15

0.25 0.50 0.1 1.0 3.0 2.5 2.5 2.0 3.5 2.3 5.8 5.0

56.0 49.0 80.0 33.4 1.0 1.1 2.1 26.0

Cl.0 Cl.0 7.5 19.6 13.5 11.3 Cl.0 Cl.0

<l.O Cl.0 4.7 15.1 <l.O Cl.0 6.0 6.0

28.0 35.3 Cl.0 <l.O 1.6 3.2 Cl.0 <l.O

TABLE III Effects of compounds 5-7 on the growth of C. fasciculata and on the sterol content of the nonsaponifiable lipids

InhibitoP

Inhibitor concentration (PM) Wet weight of cells (g) Mass of isolated sterols (mg) (ap-

proximate) Distribution of sterols (%)

Ergosterol (sterol C) 14a-Methylcholesta-8,24-dien-3&

01(A) Cholesta-8,24-dien-3/3-ol (B) Ergost-7-en-3@-ol (D) Ergo&a-7,24(28)-dien-3@-ol (E) Ergosta-5,7,22,24(28)-tetraen-3P-

01 WI Cholesta-5,7,24-trien-So-01 (G) Other

None

0.0 4.2 6.0

75.2 5.2

6.1 4.5 2.7 6.7

<l.O Cl.0

Compound 6 Compound 6 Compound 7

0.13 f 0.04 z-20 0.3 f 0.1

0.1 0.5 10.0 0.25 0.5 1.8 1.3 3.5 1.9 1.2 3.5 0.9 6.5 2.0 0.9

59.7 19.1 83.0 33.7 31.0 4.0 29.2 8.0 12.1 12.4

2.3 8.5 Cl.0 15.2 19.1 10.5 Cl.0 5.0 <l.O Cl.0

9.2 9.1 1.0 9.0 11.4 13.0 31.8 3.0 30.0 26.0

Cl.0 Cl.0 <l.O <l.O Cl.0 <l.O Cl.0 Cl.0 <l.O Cl.0

a Cont. (PM) to give 50% growth inhibition (I&.

from l-liter cultures of C. fasciculuta grown in the presence of two different concentrations of each of the inhibitors, with one of the concentrations chosen to be near the observed I50 values for each. These particular analyses, as well as the inhibitor-free control, were conducted on cultures inoculated with identical quantities of the protozoan and grown at the same time under the same conditions. They closely parallel the results from similar prior experiments. In the presence of the heteroatom-containing compounds 2 and 3, ergosterol synthesis was largely suppressed, and a new component ap- peared in the sterol mixtures (component G, RRT = 1.68). This material was subsequently isolated from cultures treated with 3 and fully characterized as cholesta-5,7,24-trien-3/3-ol (see below), which is the product expected to accumulate if the C-24 alkylation reaction was inhibited. In the presence of the cyclopropane-containing stero14, and the 22- and 23-azaster- 01s 5 and 7 the distribution of sterols was entirely different. Ergosterol synthesis was again suppressed, but the amounts of components A, B, E, and/or F were significantly increased. The precise cause(s) of these effects are less clear, but they are addressed under the “Discussion“ in the context of the probable pathways of ergosterol biosynthesis in C. fasciculata.

Isolation and Characterization of Ergosterol (1, sterol C) from C. fusciculata-The total sterols isolated from an untreated l-liter culture of C. fa.sciculata were chromatographed

on a plate of Silica Gel GF impregnated with 20% AgN03 (solvent 3:l hexane/ethyl acetate). The band with RF = 0.34 was scraped off the plate and eluted with ether-methanol to give ergosterol (2 mg, >95% pure by GC of the TMS ether, RRT = 1.45), which exhibited a chromatographic mobility, ‘H NMR spectrum, and mass spectrum identical to an authentic sample of ergosterol.

Isolation and Characterization of 14a-Methyl-5a-cholesta- 8,24-dien-3/I 01 (A)-The total sterols isolated from an untreated 2-liter culture of C. fasciculutu were chromatographed on a plate of Silica Gel GF impregnated with 20% AgN03 (solvent 191 chloroform/acetone). The band with RF = 0.58 was scraped off the plate and eluted with chloroform to give 14a-methyl-5Lu-cholesta-8,24-dien-3&ol (0.7 mg, >95% pure by GC of the TMS ether, RRT = 1.26). ‘H NMR 6 5.10 (t, lH, J = 7 Hz, 24-H), 3.62 (m, lH, 3~H), 1.68 and 1.60 (s’s, 6H, 26-H3 and 27-HJ, 0.94 (s, 3H, 19-Hs), 0.93 (d, 3H, J = 7 Hz, 21-H&, 0.88 (s, 3H, 32-H3 [14a-methyl group]), 0.71 (s, 3H, 18-HZ); MS, m/z 398 (M+, 44%), 383 (M-CH3, loo), 273 (12), 271 (9), 69 (96). Exact mass 398.3559, calculated for C28H460 398.3548. 14cY-Methyl sterols which lack 4-methyl groups are very unusual (6), but we had no authentic sample to confirm the assignment. However, a sample of 3P-acetoxy- 14cY-methyl-5cY-cholest-8-ene, which had been previously prepared by one of us (19), was hydrolyzed to give 14cY-methyl-


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5a-cholest-8-en-3P-01. The ‘H NMR spectrum of this material was extremely similar to that of sterol A (the absence of the As4 double bond would have little effect on most of the resonances), and in particular both samples showed the same characteristic methyl singlets at 6 0.71, 0.88, and 0.94. Thus, the assignment of the structure of sterol A is strongly supported.

Isolation and Characterization of Cholesta-8,24-dien-3@-ol (B) from C. fasciculata Grown in the Presence of Compound 4-The sterols from a l-liter culture of C. fusciculata treated with 0.5 pM 4 were chromatographed on a plate of Silica Gel GF impregnated with 20% AgNOa (solvent 19:l chloroform/ acetone). The band with RF = 0.54 was scraped off the plate and eluted with chloroform to give a mixture of sterols A and B in an approximately 3:2 ratio (as judged by GC analysis of the TMS ethers). Although we were unable to cleanly separate these two compounds, component B cochromatographed with authentic cholesta-8,24-dien-3@-ol (zymosterol), and the ‘H NMR spectrum of the mixture was well reproduced by a sum of the spectra of cholesta-8,24-dien-3@-ol (isolated from yeast (16)) and 14~-methyl-5a-cholesta-8,24-dien-3~-ol (A).

with 0.01 pM 3 were chromatographed on a plate of Silica Gel GF impregnated with 20% AgN03 (solvent 3:l hexane/ethyl acetate). The band with R,v = 0.30 was scraped off the plate and eluted with ether-methanol to give pure cholesta-5,7,24- trien-36-01 (G). ‘H NMR 6 5.58 (m, lH, 6-H), 5.39 (m, lH, 7- H), 5.10 (t, lH, J= 7 Hz, 24-H), 3.65 (m, 3H, 3a-H), 1.69 and 1.61 (s’s, 6H, 26-H, and 27-H& 0.94 (s, 6H, 19-Ha and 21- HZ), 0.62 (s, 3H, 18-HJ [lit. (20) (partial data) 6 1.69 and 1.63 (26-H3 and 27-HZ), 0.96 (19-HZ and 21-H3), 0.62 (18-Hs)]; MS, m/z 382 (M’, 96%), 349 (M-HZO-CHZ, loo), 323 (54), 250 (28), 210 (38), 143 (54).

Isolation and Characterization of Ergost-7-en-3/3-ol CD)-- The total sterols from an untreated lo-liter culture of C. fasciculata were chromatographed on a plate of Silica Gel GF (Analtech Uniplate Taper Plate, solvent 1:l benzene/ether) and the band of RF = 0.64 was scraped off the plate and eluted with chloroform to give 26 mg of sterols. A portion of this material (14 mg) was chromatographed on a plate of Silica Gel GF impregnated with 20% AgN03 (solvent 1:l benzene/ ether). Two bands were scraped off the plate and eluted with chloroform/methanol. The more polar fraction (RF = 0.33, 6 mg) proved to be ergosterol. The less polar fraction (RF = 0.49, 1 mg) was shown by GC analysis (of the TMS ether) to contain predominantly sterol D (RRT = 1.56) with minor contamination from sterols B and E. The ‘H NMR spectrum, mass spectrum, and chromatographic mobility of this material were essentially identical to an authentic sample of ergost-‘I- en-3/3-01.

Isolation and Characterization of Ergosta-7,24(28)-dien-38- 01 (E) and Ergosta-5,7,22,24(28)-tetraen-3P-ol (F) from C. fasciculata Grown in the Presence of Compound 7-A mixture of sterols obtained from a l-liter culture of C. fasciculata grown in the presence of 0.5 pM 7 was chromatographed on a plate of Silica Gel GF impregnated with 20% AgN03 (solvent 19:l chloroform/acetone). Two fractions with RF values of 0.51 and 0.21 were isolated. GC analysis of the less polar fraction (as the TMS ether) showed it to be sterol E (RRT = 1.70, 85% pure); similar analysis of the more polar fraction showed it to be sterol F (RRT = 1.81,73% pure (the sole contaminant was ergosterol)). The structural assignments were based on the following spectroscopic data. Sterol E: ‘H NMR 6 5.18 (m, lH, 7-H), 4.71 and 4.67 (s’s, 2H, 28-HZ), 3.62 (m, lH, 3~ H), 1.02 and 1.01 (d’s, 6H, J = 7 Hz, 26-HZ and 27-H3), 0.79 (s, 3H, 19-Ha), 0.53 (s, 3H, 18-HJ; MS, m/z 398 (M’, 76%), 383 (M-CHS, 46), 313 (22), 306 (20), 273 (32), 271 (93), 69 (100). Exact mass 398.3546, calculated for C28H460 398.3548. Sterol F: ‘H NMR d 5.95 (d, lH, J = 20 Hz, 23-H), 5.62 (dd, lH, J = 20, 8 Hz, 22-H), 5.57 (m, lH, 6-H), 5.38 (m, lH, 7- H), 4.86 and 4.83 (s’s, 2H, 28-HZ), 3.65 (m, lH, 3a-H), 1.08 (d, 6H, J = 7 Hz, 26-HZ and 27-H3), 0.94 (s, 3H, 19-H3), 0.65 (s, 3H, 18-HZ); MS, m/z 394 (M+, 61%), 361 (M-CHs-H20, 49), 253 (20), 251 (22), 123 (40), 95 (33), 81 (69), 69 (100). Exact mass 394.3221, calculated for C28H420 394.3235.

Isolation and Characterization of Cholesta-5,7,24-trien-3/3- 01 (G) from C. fasciculata Grown in the Presence of Compound 3-The sterols from a l-liter culture of C. fasciculata treated

DISCUSSION

Chromatographic analyses of the sterols of C. fasciculuta consistently showed six components. Three of these components, 14a-methyl-5a-cholesta-8,24-dien-3/3-o1 (A, Fig. 4), ergosterol (C), and ergost-7-en-3/3-ol (D), were easily isolated in pure form from cultures of the protozoa grown in the absence of any inhibitors, and they were characterized by high field proton NMR spectroscopy, low and high resolution mass spectrometry, and comparison to authentic samples or closely related molecules. Ergosterol was the predominant sterol, and it represented 75-80% of the total sterol in all such cultures. Sterols A and D typically represented 5% each of the total sterols. Three other minor sterols, 5a-cholesta-8,24-dien-3@- 01 (B, zymosterol), ergosta-7,24(28)-dien-3/3-ol (E), and ergosta-5,7,22,24(28)-tetraen-3@-ol (F), were present in untreated cultures at levels comparable to those of sterols A and D, but their chromatographic properties made them more difficult to isolate in pure form. The spectroscopic samples of sterols B, E, and F were obtained from samples of C. faxiculata cultured in the presence of inhibitors which caused these sterols to accumulate.

Fig. 4 shows the likely biosynthetic relationships between the sterols isolated from C. fasciculata. The biosynthesis of ergosterol has been extensively investigated in yeast and fungi, and the modification of the side chain, which is of greatest interest to us, occurs at different stages in the overall process in different organisms (21-22). In most fungi, the side chain alkylation occurs on C& sterol precursors of ergosterol, but in yeast the side chain is methylated only after demethylation at C-4 and C-14 to give CZ7 sterols (21). The spectrum of sterols isolated from C. fasciculata indicates that, in general, the yeast pathway is followed in this protozoan, and this

FIG. 4. Probable pathways of sterol biosynthesis in C. fasciculuta. Only those sterols which have been isolated from C. fa.sciculata are lettered. Solid arroa~.s indicate transformations that should require only one enzymatic step; dashed arrows indicate transformations that require more than one reaction. The transformation B-G appears to occur only in the presence of inhibitors 2 and 3.


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result is consistent with similar findings in Trypanosoma cruzi (3), and various strains of Leishmania (5).

The finding of 14~-methyl-5ol-cholesta-8,24-dien-3&ol (A) in substantial quantities in C. fasciculata is quite significant. 14a-Monomethyl sterols (which lack 4-methyls) are very rare (6, 32-35), and, for example, they have never been observed in mammals, although 4cu-methyl and 4,4-dimethyl sterols are common. For this reason it is usually concluded that the oxidative demethylation of CSO sterols begins in most organisms with removal of the 14cu-methyl group followed by removal of the two 4-methyl groups. However, the isolation of 14~-methyl-5a-cholesta-8,24-dien-3P-o1 from ketoconazole treated Lekhmania mexicana mexicana has also been reported (34), and thus it may be that in C. fasciculata and other trypanosomatids the order of methyl group removal is re- versed or that there is no priority for attack at either site. If the unusual order of demethylation should prove to be a strict requirement and common to other trypanosomatids, then this metabolic anomaly might be exploited in the design of other protozoan-specific inhibitors of sterol biosynthesis.

In the present study, all of the compounds which were tested for effects on growth and sterol biosynthesis in C. fasciculata were designed to interfere with one or more steps in ergosterol side chain synthesis. We chose to prepare our potential inhibitors as derivatives of cholestanol, in the hope that the fully saturated steroid nucleus would not undergo as rapid metabolic processing as more highly functionalized sterols. (We have no data at present concerning the metabolism of the inhibitors themselves, but it is worth noting that a great many cholestenols, cholestadienols, and assorted steroid diols and triols have been shown to be convertible to cholesterol in animal tissues or cell-free preparations (6), even though many or most of these compounds cannot have a significant role in normal sterol biosynthesis.) Five of the six compounds tested were potent inhibitors of the growth of C. fasciculata at submicromolar concentrations in the culture medium (see Tables II and III); only the 23-thiacholestanol (6) was without effect.

The two 24-heteroatom-substituted sterols 2 and 3, which exhibited IbO values of 0.32 pM and 9 nM, respectively, ap- peared to have similar modes of action. When the sterol distributions in protozoa treated with these inhibitors were examined, it was found that ergosterol synthesis was largely suppressed, and that the principal sterol observed was cholesta-5,7,24-trien-3/l-ol (G), a sterol which is not observed in untreated cultures of C. fasciculata (see Table II). In the presence of 50 nM 3 in the culture medium, almost 90% of the sterol found in the protozoa was G. It is clear from the presence of the A5, A7, and AZ4 double bonds in sterol G that compounds 2 and 3 block the alkylation at C-24 of the sterol side chain but do not interfere with the normal enzymatic processing of the steroid nucleus. The fact that no CZ7 sterols containing a A” double bond were observed in this experiment (or any other) suggests that C-22,23 desaturation cannot occur in C. fasciculata in the absence of an alkyl group at C-24. In other organisms C-22,23 desaturation in unsubstituted side chains is rare (23), but in Oehlschlager et al.‘s (9) study of AZ4-methyltransferase inhibitors in yeast, substantial accu- mulations of cholesta-5,7,22,24-tetraen-3/3-ol were observed when cultures were treated with various 25-azasterols, dem- onstrating that C-22,23 desaturation had occurred in a C27 sterol.

The azastero13, which is positively charged at physiological pH, is presumably an analog of a carbonium ion intermediate in the A24-methyltransferase reaction. Such a reaction intermediate analog should bind tightly at the active site of the

methyltransferase (24), and this concept is generally invoked to explain the observed inhibition of many carbonium ion- utilizing enzymes by positively charged substrate analogs (8- 11,25,26). However, the thiastero12 is not positively charged, yet it is also a potent inhibitor of the C-24 alkylation. We hypothesize that the thioether is methylated in the active site of the AZ4-methyltransferase to yield a sulfonium ion which then functions as a cationic reaction intermediate analog in the usual way. We have previously shown that several thioether-bearing fatty acids are potent inhibitors of the biosynthesis of dihydrosterculic acid (a cyclopropane-containing fatty acid) in C. fasciculata (8, 27). This cyclopropane synthesis is another example of an S-adenosylmethionine- dependent methyl transfer to an olefin, and it is likely that a similar mechanism of inhibition, methylation of the thioether to give a sulfonium ion at the active site, is involved as well (1%

SCHEME 2

The sterol distributions in C. fasciculata treated with 22- azacholestanol (5) and 23-azacholestanol (7) were entirely different from those discussed above. Ergosterol synthesis was suppressed, but no cholesta-5,7,24-trien-3/3-ol was observed in either case. Instead, the principal sterol to accumulate was ergosta-5,7,22,24(28)-tetraen-3P-ol (F), and somewhat elevated levels of other minor sterols were also observed (see Table III). Conversion of sterol F to ergosterol requires only the reduction of the A 24(28) double bond, and thus inhibition of the A24’28’-reductase is the most likely site of inhibition by compounds 5 and 7. In support of this conclusion, the amount of ergost-7-en-3P-ol (D) was also observed to be greatly reduced relative to control experiments, and the levels of its presumed precursor, ergosta-7,24(28)-dien-3p-ol (E), were substantially elevated. At a high concentration (0.5 pM) compound 5 also produced a substantial accumulation of sterol A, suggesting that there is at least one other site of inhibition; however, we have no simple explanation for this effect.

Interestingly, 23-thiacholestanol (6), unlike 23-azacholestanol, had no significant effect on the sterol distribution in C. fasciculuta. This observation provides some degree of support for our suggested mechanism of methyltransferase inhibition by 24-thiacholestanol (2). All of the azasterols will be positively charged at physiological pH, and this charge is probably required for them to act as inhibitors. However, compound 2 only can become charged by methylation of the sulfur, since thioethers are not significantly basic. In compound 6, the sulfur at position 23 is poorly situated to be methylated by an enzyme that normally acts on carbon-24. Thus, 6 is never methylated, it is never charged, and it has no inhibitory effect.

The most unusual inhibitor was the biscyclopropyl sterol 4. The C-22,23 desaturation is likely to be initiated by a hydrogen atom abstraction to give a carbon radical at either C-22 or C-23. We hoped that by placing cyclopropanes adja- cent to these sites, an intermediate cyclopropylcarbinyl radical, if formed, would undergo the well known rearrangement (28, 29) to a homoallyl radical which might then react with some active site group to inactivate the enzyme. This strategy has proven successful in developing inhibitors of a number of oxygenases (30, 31). However, the sterol distribution in C.


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fasciculata treated with compound 4 was inconsistent with inhibition of any single reaction. Increased amounts of sterols A, B, E, as well as greatly reduced amounts of ergosterol and sterol D were observed, but these results are not easily interpreted.

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M D Rahman and R A Pascal, JrCrithidia fasciculata.

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan

1990, 265:4989-4996.J. Biol. Chem.

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