Biochimica et Biophysica Acta, 1040 (1990) 95-101
95
Elsevier BBAPRO 33704
Non-sterol structural probes of the lanosterol 14a-demethylase from S a c c h a r o m y c e s cerevisiae * G e r a r d D . W r i g h t , T o d d P a r e n t a n d J o h n F. H o n e k Guelph-Waterloo Centerfor Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo (Canada)
(Received 16 January 1990) (Revised manuscript received3 April 1990)
Key words: Lanosteroldemethylase;Non-sterolprobe; (S. cereoisiae) A number of non-sterol iron-liganding molecules were used to probe the active site of the lanosteroi 14a-demethylase from Saccharomyces cerevisiae. Simple bi- and tricyclic aromatic amines were found to exhibit Type II binding spectra with the demethylase. Stereochemical and positional effects appear to play critical roles in the binding of these compounds to the demethylase. These compounds have been used to generate additional active-site structural information on this enzyme, currently a target for the development of new antifungal agents.
Introduction Systemic mycotic infections have become significant clinical causes of morbidity over the last decade. Much attention is currently being focused on fungal pathogens in man largely due to the advent of transplant surgery and its concomitant use of immunosuppresive drugs, corticosteroid hormones and broad-spectrum antibiotics, conditions which markedly alter man's resistance to infection by fungi [1]. One fungal enzyme, the lanosterol 14a-demethylase, is considered a potential target enzyme for drug design. This cytochrome P-450 enzyme, which oxidatively removes the 14a-methyl group in lanosterol to produce 4,4-dimethyl-5acholesta-8,14,24-triene-3fl-ol and formic acid (Fig. 1), plays a key role in the biosynthetic pathway to ergosterol, a major fungal sterol. Several clinically utilized antimycotic agents such as ketoconazole, fluconazole and others appear to function by ligation of their imidazole or triazole nitrogen to the heme-iron of this demethylase, preventing the conversion of lanosterol to the corresponding triene [2]. The resulting alteration in ergosterol concentration in fungal membranes may
affect membrane permeability and the activities of membrane-associated enzymes such as chitin synthase [3]. Although much effort has been expended on the development of imidazole and triazole based inhibitors of this enzyme, little fundamental biochemical information is available concerning the active site of the enzyme [4]. Presently, the only crystal structure of a cytochrome P-450 determined to date is the camphor hydroxylase from Pseudomonas putida [5]. Although there appears to be some sequence homologies within the cytochrome P-450 superfamily, the active site structure will vary for each P-450. In order to gain further insight into the structure of the active site of the fungal demethylase, the enzyme from a Saccharomyces cerevisiae clone containing the yeast lanosterol 14a-demethylase on an expression plasmid [6] was partially purified and the active site was methodically probed by investigating the binding affinities of various liganding probes with the enzyme. This demethylase has high amino acid sequence homology to the recently reported lanosterol demethylase sequences from the pathogenic yeasts Candida albicans [7] and Candida tropicalis [8].
* Dedicated to the memoryof Dr. Bernard Belleau. Abbreviations: DMF, dimethylformamide; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; MIC, minimum inhibitory concentration. Correspondence: J.F. Honek, (GWC) 2, Department of Chemistry, Universityof Waterloo, Waterloo,Ontario, N2L 3G1 Canada.
oNAoPH Fig. 1. Reaction catalyzedby the lanosterol 14a-demethylasefrom S.
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
cerevisiae.
96 Materials and Methods Compounds 1, 5, 24 and 27 were from J.T. Baker Chemicals (Phillipsburg, N J). Compounds 2, 6, 7, 8, 12, 13, 15, 25, 26, 6-methoxy-l-tetralone, 1,2,3,4-tetrahydrophenanthrene-l-one, 2-naphthaldehyde, 1-naphthylacetonitrile, 2-naphthylacetonitrile and phenanthrene-9carboxaldehyde were from Aldrich (Milwaukee, WI). 1,8-Dimethyhiaphthalene was from Fluka (Ronkonkoma, NY). Compound 23 was from Sigma (St Louis, MO). All other chemicals were of the highest quality available. S. cerevisiae JL745/pVK1 was the generous gift of Dr. J. Loper, University of Cincinnati.
Purification of S. cerevisiae P-450 P-450 was partially purified as follows. Typically, 15 1 of a 24 h culture of S. cerevisiae JL745/pVK1 grown in YEPD medium (0.5% yeast extract, 0.5% tryptone, 0.5% potassium phosphate, 3% glucose, pH 5.5) was harvested in a Sharpies continuous flow centrifuge. Cells were washed with 1.0 M sorbitol and disrupted by glass bead homogenization (Bead Beater, Biospec Prod., Bartsville, OK) for a total of 5 min (cooled in NaCl-ice slurry) (30 s disruption at 30 s cooling intervals). Cell debris was removed by centrifugation (3000 × g for 10 min) and poly(ethylene glycol) (8000 MW) was added to give a final concentration of 7.5% (w/v). Microsomes were isolated by centrifugation at 30 000 x g for 25 min. P-450 was extracted from the microsomes as described [9]. The solubilized material was applied to an octylamino Sepharose 4B column (100 ml) [101 equilibrated with 100 mM phosphate, 1 mM EDTA, 0.5~ sodium cholate and 20% (v/v) glycerol (pH 7.0) at 4 ° C. The column was washed with 2 vols. of equilibration buffer and the P-450 eluted with 100 mM phosphate, 1 mM EDTA, 0.2% (v/v) Tergitol 15-S-12 and 20% (v/v) glycerol (pH 7.0). P-450 containing fractions were pooled and concentrated over an Amicon PM30 ultrafiltration membrane to 0.5 /~M. P-450 was typically recovered in 80% final yield and with a specific content of 0.10 mnol P-450/mg protein. Spectral titrations Compounds were dissolved in ethanol (which has no detectable effect on P-450 at these concentrations) and added to a cuvette containing 800/~1 of partially purified P-450, an equal amount of ethanol was added to the reference cuvette also containing enzyme. The difference spectrum was recorded from 350-500 rim. The absorbance difference between 426 and 408 was appropriately plotted versus compound concentration using the Enzfitter (Elsevier) software to evaluate binding constants (Scatchard analysis, one binding site).
Synthesis of compounds Unless specified, all amines were taken up in diethyl ether and precipitated as their HC1 salts by passage of dry HC1 gas for 30 min for ease in purification. Physical data reported are for the isolated hydrochlorides. 1HNMR spectra were obtained on a Bruker AC200 (200 MHz) or AM250 (250 MHz) instrument. Mass spectra were recorded using chemical ionization (NH 3 as carrier gas), on a VG ZAB-E instrument by Dr. R.W. Smith (McMaster University, Hamilton, Ontario). Compound 3 was obtained in 6% yield by reductive amination of 6-methoxy-l-tetralone with sodium cyanoborohydride in the presence of ammonium acetate [11]. mp: 253-270°C dec. 1H-NMR (free base in CDC13): ~ 7.31 (d, 1H), 6.75 (dd, 1H), 6.60 (d, 1H), 3.95 (t, 1H), 3.79 (s, 3H), 2.75 (m, 2H), 1.95 (m, 2H), 1.70 (m, 2H), 1.55 (s, 2H, exchanges with D20 ). MS: m / z (relative intensity), 177 (M, 1.7%, 176 (5%), 161 (100%). Compound 20 was obtained similarly from 1,2,3,4-tetrahydrophenanthrene-l-one in 23% yield, mp: > 255°C. "H-NMR (free base in CDC13): 8 8.0 (d, 1H), 7.78 (d, 1H), 7.67 (d, 1H), 7.48 (m, 3H), 4.10 (t, 1H), 3.13 (m, 2H), 2.0 (m, 2H), 1.8 (m, 2H), 1.59 (s, 2H, exchanges with D20 ). MS: m / z (relative intensity), 198 ( M + 1, 2.2%), 181 (100%). 6-Methoxy-l-tetralone was homologated to the corresponding 1-carboxaldehyde by treatment with trimethvisulfoxonium iodide and sodium hydride as previously described [12]. Compound 4 was obtained in 6% overall yield by conversion of the aldehyde to the oxime with hydroxylamine hydrochloride in pyridine/ethanol followed by reduction with sodium borohydride in the presence of TiC14 [13] mp: 195-207°C dec. 1H-NMR (free base in CDC13): 8 7.12 (d, 1H), 6.72 (dd, 1H), 6.61 (d, 1H), 3.78 (s, 3H), 2.93 (m, 2H), 2.7 (m, 3H), 1.80 (m, 4H), 1.39 (s, 2H, exchanges with D20 ). MS: m / z (relative intensity), 192 ( M + 1, 100%), 175 (31%), 161 (38%). Similarly, compound 21 was obtained in 9% overall yield from 1,2,3,4-tetrahydrophenanthrene-l-one. rap: > 2 6 0 ° C begins to decompose at 254°C. 1H-NMR (d6DMSO): 6 8.1 (br s, 2H, exchanges with D20 ), 8.03 (d, 1n), 7.89 (d, 1n), 7.67 (d, 1H), 7.5 (m, 2H), 7.46 (d, 1H), 3.18 (m, 3H), 3.0 (m, 2H), 1.89 (m, 4H). MS: m / z (relative intensity), 212 ( M + 1, 100%), 195 (7.4%), 182 (13.4%). Compound 9 was prepared from the corresponding nitrile by reduction with sodium borohydride in the presence of CoC12 [14]. mp: 184-188°C. aH-NMR (d6DMSO): 8 8.25 (br s, 2H, exchanges with D20 ), 8.16 (d, 1n), 7.96 (d, 1H), 7.87 (d, 1U), 7.56 (m, 2I-I), 7.48 (m, 2H), 3.43 (m, 2H, becomes a triplet upon addition of D20 ), 3.11 (m, 2H). Compound 11 was similarly obtained in 25% yield from the appropriate nitrile, nap: 254-257 °C decomposes to a brown semi-solid. 1H-NMR (d6DMSO): 8 8.28
97 (br s, 2H, exchanges with D20 ), 7.88 (m, 3H), 7.76 (s, 1H), 7.50 (m, 3H), 3.15 (s, 4H, becomes a multiplet upon addition of 1)20). Compound 10 was prepared from the corresponding oxime (prepared from the commercially available aldehyde was described above) by reduction with LiA1H 4 in THF in 59% yield, mp: > 260°C begins to decompose at 245 ° C. 1H-NMR (d6DMSO): 8 8.73 (br s, 2H, exchanges with D20), 8.04 (s, 1H), 7.92 (m, 3H), 7.69 (dd, 1H), 7.55 (m, 2H), 4.20 (s, 2H). Compound 14 was obtained by bromination of 1,8dimethylnaphthalene with N-bromosuccinimide followed by treatment with 2,2,2-trifluoroacetamide and Nail in DMF to give the protected cyclic amine as previously described [15]. Hydrolysis of the amide with sodium carbonate in aqueous methanol followed by precipitation of the hydrochloride with HC1 in diethyl ether gave 14 in a 23% overall yield, mp: > 260°C, begins to brown at 170°C. aH-NMR (d6DMSO): 8 9.9 (br s, 1H, exchanges with D20 ), 7.95 (d, 2H), 7.55 (m, 4H), 4.66 (s, 4H). MS: m / z (relative intensity), 170 ( M + 1, 100%), 155 (6%). Reduction of the oxime obtained from phenanthrene-9- carboxaldehyde with NaBH 4 in the presence of TiC14 gave 16 in a 48% overall yield, mp: 246-252°C (decomposes), 1H-NMR (d 6 DMSO): 8 8.70 (m, 2H), 8.50 (br s, 2H, exchanges with D20), 7.99 (m, 1H), 7.82 (m, 2H), 7.5 (m, 4H), 4.4 (d, 2H, becomes a singlet upon addition of D 2 0 ). M S : m / z (relative intensity), 208 ( M + 1, 64%), 191 (100%). Reduction of phenanthrene-9-carboxaldehyde with sodium borohydride in methanol afforded the alcohol in a > 95% yield. Treatment with Lawson's reagent in toluene [16] gave 18 in 19% yield, mp: 82-84°C. 1HNMR (CDC13): 8 8.72 (m, 2H), 8.25 (m, 1H), 7.65 (m, 6H), 4.25 (d, 2H), 1.9 (t, 1H). MS: m / z (relative intensity), 224 (M, 39%), 191 (100%), 165 (9.7%). The alcohol could be coverted to the chloride by the method of Corey, Kim and Takedu [17] in 75% yield. Reaction with imidazole in DMF gave compound 19 in a 84% yield, mp: 118-120°C. ~H-NMR (CDC13): 8.76 (d, 1H), 8.68 (d, 1H), 7.91 (d, 1H), 7.80 (d, 1H), 7.65 (m, 5H), 7.40 (s, 1H), 7.12 (s, 1H), 6.99 (s, 1H), 5.62 (s, 2H). MS: m / z (relative intensity), 259 ( M + 1, 100%), 191 (21%), 69 (42%). Displacement of the chloride with sodium cyanide in DMF followed by reduction with LiA1H 4 in diethyl ether gave compound 17 in a 26% yield, mp: > 260°C, begins to decompose to a brown solid at 210 ° C. 1HNMR (d6DMSO): 8 8.89 (m, 2H), 8.20 (m, 1H), 7.98 (m, 3H, becomes 1H after addition of D20 ), 7.70 (m, 5H), 3.3 (m, 4H, becomes two triplets at 8 3.40 and 3.22 with the addition of D20 ). MS: m / z (relative intensity), 222 ( M + 1, 100%), 192 (21%). Compound 22 was prepared from 1-methylphenanthrene which was obtained by reaction of 1,2,3,4-tetra-
hydrophenanthrene-l-one with methylmagnesium bromide followed by dehydrogenation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. 1-Methylphenanthrene was brominated with N-bromosuccinimide and treated with hexamethyltetramine as described [18] to give 22 in a 16.5% overall yield, mp: > 260°C, begins to brown at 244°C., 1H-NMR (d6DMSO): 8 8.5 (m, 2H), 8.38 (br s, 2H, exchanges with D20), 8.07, (m, 3H), 7.75 (m, 4H), 3.19 (2, 2H, becomes a singlet upon addition of D20 ). MS: m / z (relative intensity), 208 (M + 1, 100%), 191 (79%).
Determination of MIC MICs were determined by the method of Gordon et al. [19] using final concentrations of test compounds of 500, 250, 100, 75, 50, 25, 10, 5 and 1 /xM. Tests were performed against the yeasts S. cerevisiae Y222 and S. cerevisiae 239 whose P-450 content has been previously described [20]. Results
Table I summarizes the K s or binding affinity for the various amine, imidazole and thiol liganding compounds (Fig. 2) as determined by analyses of the difference spectra generated by the yeast demethylase upon interaction with the compounds. The K s determined is a good measure of the affinity of these molecules for yeast P-450 as none of the compounds studied showed
TABLE I
Binding affinities of various probes on S. cerevisiae lanosterol demethylase Assays were conducted as outlined in Materials a n d Methods. C o m p o u n d No.
K s (#M)
C o m p o u n d No.
K s (#M)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
N.I. N.I. N.I. N.I. 331+260 112+36 a 53+9 209 5:93 505:11 580 5:423 N.I. 308 5:98 14+4
15 16 17 18 19 20 21 22 23 24 25 26 27
b
N.I., no interaction. a Some interaction, c o m p o u n d spectrum interferes. b N o interaction at 37 # M . c Strong interaction. d N o interaction at 157/~M. e N o interaction at 187 # M . r N o interaction at 276 # M .
21 + 19 N.I. c 22+2 N.I. 841 + 466 22+6 N.I. 2.6 m m s : 1.4 d e r
98
CL
NH2
~ N H
,•R
2
CH30 1
2
3, R: NH 2 4, R : CHzNH 2
{ ~NH
~ N H
2
2
6
5
7, R : NH2 8, R = CH2NH2 9, R : CH2CH2NH 2
J~ 10, 11,
R=CH2NH 2 R=CHzCH2NH 2
12, (R) 13, (S)
N H 14
]'vN HZ R
R : NH2 16, R = CH2NH;~ 17, R =CH2CH2 NH 2 18, R= CH2SH
15,
19, R=
22
20, R: NH 2 21, R =CH2NH2
CH2N,~N
0
NH2 24
23
CH3 0 . ~ H 25,
(R)
27
26, (S)
Fig. 2. Chemical structure of c o m p o u n d s utilized in this investigation.
99 which binding data could not be obtained (Fig. 4), however, this molecule appeared to show high affinity for the demethylase based on the extent of concentration-dependent spectral change. The insolubility of several of the compounds precluded a detailed study of their binding to the enzyme, however, these compounds and the highest concentrations at which little or no binding was observed are as follows: 1 (2.8 mM), 2 (1.5 mM), 3 (1.8 raM), 4 (270 #M), 12 (400 #M), 17 (80 #M), 20 (1 mM) and 23 (93 #M). In addition, a-naphthylamine 7 showed some interaction at 100 #M and 9-aminophenanthrene 15 showed no interaction with the enzyme up to 37 #M but spectral interference prevented further investigation of these amines at higher concentrations. Octanol/water partition coefficients (logP) for each compound were calculated by the method of Hansch and Leo [23]. No correlation was found between logP and K s for the compounds tested (data not shown). Minimum inhibitory concentrations for compounds 4, 10, 11, 14, 17, 18, 19 and 21 were determined against two ergosterol containing S. cerevisiae strains, one of which, Y222, contained levels of cytochrome P-450 of 36.5 pmol/mg protein and strain 239 contained levels below detection limits [20]. Strain Y222 was only susceptible to 19 (500 #M), whereas strain 239 was susceptible to compounds 10, 17 and 21 (500 #M), 14 (250 #M) and 19 (75 #M).
o~.¢l c~a0Q8C12(t 3
g
o.4o
n O0
•
|
" ~500J
I
I
I
I
5eo
I
i
I
I
I 86o
I
I
I
I
I
I
74o I 820
5.40 620 " ZOO 7.80 a Absorbance/concentrat ion
I
l
I
--
900[ 10-4 8.60 9.40
Fig. 3. Spectral titration of the demethylase with imidazole 19. The concentration of demethylase was 0.5 /~M in 100 mM potassium phosphate containing 1 mM EDTA, 0.25g Tergitol 15-S-12 and 20% glycerol (pH 7.0), 23°C; p a t h = l cm. Analysis of the titration in which the concentration of bound and free 19 were calculated using the extent of spectral change ( K s = -slope) at the wavelengths indicated. The inset shows the difference spectra obtained following additions of 4.1, 8.1, 12.1, 16.1, 20.1 and 24 #M imidazole 19.
saturation when the concentrations of ligand and enzyme were equivalent [21]. All nitrogen containing molecules gave typical Type II difference spectra [22] (peak near 426 nm and a trough at 408 nm) as exemplified by the imidazole compound 19 (Fig. 3). The 9-thiomethylphenanthrene 18 gave complex spectra from
B
I =000 !
•
360
360
400
/*40 480 Wovelength (nm)
520
560
I
•
I
•
I
•
!
•
400 440 Wcvetength (nrn)
|
.
I
.
480
600
Fig. 4. Spectral titration of the demethylase with thiol 18. The conditions of the assay were identical to that used in Fig. 3 in all respects. (A) The absolute spectra of the enzyme following additions of 0.0, 15.4, 30.5, 45.1, 59.5, 73.5 and 87.1 # M thiol 18. (B) The difference spectra obtained under the above conditions with concentrations of 18 of 7.8, 15.4, 23.0, 30.9, 37.8, 45.1 and 52.3 #M.
100 Discussion
Although problems with solubility and spectral interference with several probes were encountered in this study, which, in some cases, may have accounted for larger errors in K s determinations, general conclusions about the structure-activity relationships of these probes may still be made. Amines attached to saturated ring systems such as compounds 1-4 and 20-21 have very little affinity for the demethylase. This might be due to unfavorable conformations of the saturated ring disallowing correct positioning of the liganding amine for interaction with the iron atom. Probes having poorly basic amines, such as the aromatic amines 5-7 and 15 exhibited moderate to weak interactions with the enzyme. The spectral interference produced by several of these probes hinders a more detailed investigation with these analogues. Interposing a carbon atom between the liganding functionality and the aromatic ring systems enhanced binding to the demethylase, as shown with compounds 8 and 10. There appears to be sufficient space in the active site of the enzyme to bind both the a- and fl-substituted naphthalene probes with similar affinities. However, the enzyme becomes more discriminating against more extended amines such as the ethylamine probes 9 and 11; the a-substituted compound 9 binding much less effectively than 8 and 10 but still more effectively than 11, a result indicating a limitation to the size and/or shape of the active site for this enzyme. As there exists free rotation around one of the bonds in compound 8, the more restricted analogue 14 was prepared and studied with the enzyme. Binding affinity for the demethylase increased for this compound perhaps indicating a more restricted but favorable orientation of the amine for interaction with the iron atom. A further delineation upon the size and/or shape of the demethylase active site is the surprisingly differential interaction observed between the two methyl homologs 12 and 13 and their parent compound 8. Incorporation of an additional methyl group proximal to the amine group in 8 results in greatly reduced binding with the S isomer and little or no binding for the R isomer. Conformational analysis (unpublished observations) indicates a differential positioning of the naphthalene moieties in space for these two compounds which could then be discriminated for by the active site of the demethylase. To evaluate the effect of an additional ring structure on the binding affinity to the enzyme, various phenanthrene probes were also synthesized and studied with the demethylase. As previously mentioned, the saturated tetrahydrophenanthrenes 20 and 21 do not bind effectively with the enzyme, a result apparently confirming the importance of aromatic character to binding affinity. A similar importance of the intervening atoms between the aromatic rings and the liganding
atom can be seen in the binding affinities for compounds 15, 16 and 22 where the additional carbon atom enhances the interaction between the enzyme and the probes. The active site appears to be sufficiently large to accommodate the phenanthrenes 16 and 22 equally well and it is possible that the phenanthrene rings of these two compounds occupy identical regions in the active site. Comparing naphthalene 8 and phenanthrene 22, the additional aryl ring increases binding affinity to the enzyme and indicates that sufficient space is present to accommodate this additional ring. A similar comment could be made concerning compounds 10 and 16. The thiol containing phenanthrene, 111, would also be predicted to bind and indeed this is observed. However, comparison of the binding to the amine analogs cannot readily be made as the spectral change is fundamentally different from that produced by the amines. Thiol containing molecules have recently been utilized to probe the cytochrome P-450 enzymes from crude rat microsomes [24]. Inclusion of an additional carbon atom between the amine and the aromatic portion reduces the binding affinity as shown for compound 17. Interestingly, the imidazole, 19, binds quite effectively to the enzyme although the distance between the liganding nitrogen and the arene is much greater than that found in 16. Restricted analogues of 19 might further clarify this interaction. Various other amines were investigated as to their interaction with the demethylase. Aminoglutethimide, 23, has been shown to interact with cytochromes P-450 involved in mammalian steroid biosynthesis [25]. This compound showed no interaction with the enzyme at 93 #M. Quinoline, 24, showed low affinity for the enzyme as exemplified by its large binding constant. Quinine, guinidine and carbazole, 25, 26 and 27 were all devoid of binding affinity to the enzyme at the concentrations investigated where solubilities were not exceeded. MIC studies performed on two yeast strains containing different amounts of cytochrome P-450 indicated that several of the compounds active in our enzyme assays inhibited cell growth. Considerations such as transport and extent of protonation may be contributing factors determining the observed inhibitory potential of these compounds. In conclusion, based on the probes evaluated in this study, a discrete binding site that can accommodate planar aromatic ligands exists for this enzyme. At the very least, the enzyme can accommodate planar aromatic compounds such as naphthalene and phenanthrene as well as the sterol lanosterol. In order to increase the binding interactions between the demethylase and the non-sterol probes discussed in this study, additional interactions expected between the normal substrate, lanosterol and the demethylase should be considered. A critical hydrogen bond between the 5-keto group in camphor and tyrosine-96 in camphor hydroxylase has
101 b e e n n o t e d [5]. B a s e d o n o u r studies w i t h l a n o s t e r o l a n a l o g u e s m o d i f i e d at the 3 - h y d r o x y l p o s i t i o n ( u n p u b lished o b s e r v a t i o n s ) a n d those r e c e n t l y r e p o r t e d b y A o y a m a a n d c o - w o r k e r s [26], a s i m i l a r i n t e r a c t i o n a p p e a r s to exist b e t w e e n the 3 - h y d r o x y l g r o u p o f l a n o s t e r o l a n d a n a m i n o a c i d side c h a i n in the active site. H y d r o x y l a t e d a n a l o g u e s w h i c h t a k e this p o t e n t i a l i n t e r a c t i o n i n t o a c c o u n t c o u l d well d i s p l a y e n h a n c e d b i n d i n g . A d d i t i o n a l h y d r o p h o b i c functionalities ( a r o m a t i c , alkyl) a p p r o p r i a t e l y p o s i t i o n e d in the a n a l o g u e s h o u l d also increase the b i n d i n g affinities o f these molecules.
Acknowledgements W e wish to t h a n k Dr. J o h n L o p e r for k i n d l y p r o v i d ing the yeast strain utilized in this work. W e g r a t e f u l l y a c k n o w l e d g e the financial s u p p o r t of N S E R C ( C a n a d a ) .
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7 Lai, M.H. and Kirsch, D.R. (1989) Nucleic Acids Res. 17, 804. 8 Chen, C., Kalb, V.F., Turi, T.G. and Loper, J.C. (1988) DNA 7, 617 -626. 9 Yoshida, Y. and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660. 10 Guengerich, F.P. (1982) in Principles and Methods od Toxicology (Hayes, A.W., ed.), pp. 609-634, Raven Press, New York. 11 Borch, R.F., Bernstein, M.D. and Durst, H.D. (1971) J. Amer. Chem. Soc. 93, 2897-2904. 12 Corey, E.J. and Chaykovsky, M. (1969) Org. Syn. 49, 78-80. 13 Kano, S., Tanaka, E., Sugino, S. and Hibinu, S. (1986) Synthesis, 695-697. 14 Satoh, T., Suzuki, S., Suzuki, Y., Miyaji, Y. and Imal, Z. (1969) Tetrahedron Lett. 52, 4555-4558. 15 Hawkins, J.M. and Fu, G.C. (1986) J. Org. Chem. 51, 2820-2822. 16 Nishio, T. (1989) J.C.S. Chem. Commun., 205-206. 17 Corey, E.J., Kim, C.U. and Takeda, M. (1972) Tetrahedron Lett. 42, 4339-4342. 18 Delepine, M. (1895) Bull. Soc. Chim. Fr. 13, 358. 19 Gordon, M.A., Lapa, E.W. and Passero, P.G. (1988) J. Clin. Microbiol. 26, 1874-1877. 20 Wright, C.D. and Honek, J.F. (1989) Can. J. Mierobiol. 35, 945-950. 21 Schuster, I. (1985) Xenobiotica 15, 529-546. 22 Jefcoate, C.R. (1978) Methods Enzymol. 52, 258-279. 23 Hansch, C. and Leo, A. (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology, J. Wiley & Sons, New York. 24 Slyn'ko, N.M., Leonova, I.N., Pak, E.V., Slepneva, I.A. and Weiner, L.M. (1988) Molecular Biology (USSR) 22, 800-804 (and references therein). 25 Shaw, M.A., Nicholls, P.J. and Smith, H.J. (1988) J. Steroid Biochem. 31, 137-146. 26 Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1989) Biochina. Biophys. Acta 1006, 209-213.
95
Elsevier BBAPRO 33704
Non-sterol structural probes of the lanosterol 14a-demethylase from S a c c h a r o m y c e s cerevisiae * G e r a r d D . W r i g h t , T o d d P a r e n t a n d J o h n F. H o n e k Guelph-Waterloo Centerfor Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo (Canada)
(Received 16 January 1990) (Revised manuscript received3 April 1990)
Key words: Lanosteroldemethylase;Non-sterolprobe; (S. cereoisiae) A number of non-sterol iron-liganding molecules were used to probe the active site of the lanosteroi 14a-demethylase from Saccharomyces cerevisiae. Simple bi- and tricyclic aromatic amines were found to exhibit Type II binding spectra with the demethylase. Stereochemical and positional effects appear to play critical roles in the binding of these compounds to the demethylase. These compounds have been used to generate additional active-site structural information on this enzyme, currently a target for the development of new antifungal agents.
Introduction Systemic mycotic infections have become significant clinical causes of morbidity over the last decade. Much attention is currently being focused on fungal pathogens in man largely due to the advent of transplant surgery and its concomitant use of immunosuppresive drugs, corticosteroid hormones and broad-spectrum antibiotics, conditions which markedly alter man's resistance to infection by fungi [1]. One fungal enzyme, the lanosterol 14a-demethylase, is considered a potential target enzyme for drug design. This cytochrome P-450 enzyme, which oxidatively removes the 14a-methyl group in lanosterol to produce 4,4-dimethyl-5acholesta-8,14,24-triene-3fl-ol and formic acid (Fig. 1), plays a key role in the biosynthetic pathway to ergosterol, a major fungal sterol. Several clinically utilized antimycotic agents such as ketoconazole, fluconazole and others appear to function by ligation of their imidazole or triazole nitrogen to the heme-iron of this demethylase, preventing the conversion of lanosterol to the corresponding triene [2]. The resulting alteration in ergosterol concentration in fungal membranes may
affect membrane permeability and the activities of membrane-associated enzymes such as chitin synthase [3]. Although much effort has been expended on the development of imidazole and triazole based inhibitors of this enzyme, little fundamental biochemical information is available concerning the active site of the enzyme [4]. Presently, the only crystal structure of a cytochrome P-450 determined to date is the camphor hydroxylase from Pseudomonas putida [5]. Although there appears to be some sequence homologies within the cytochrome P-450 superfamily, the active site structure will vary for each P-450. In order to gain further insight into the structure of the active site of the fungal demethylase, the enzyme from a Saccharomyces cerevisiae clone containing the yeast lanosterol 14a-demethylase on an expression plasmid [6] was partially purified and the active site was methodically probed by investigating the binding affinities of various liganding probes with the enzyme. This demethylase has high amino acid sequence homology to the recently reported lanosterol demethylase sequences from the pathogenic yeasts Candida albicans [7] and Candida tropicalis [8].
* Dedicated to the memoryof Dr. Bernard Belleau. Abbreviations: DMF, dimethylformamide; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; MIC, minimum inhibitory concentration. Correspondence: J.F. Honek, (GWC) 2, Department of Chemistry, Universityof Waterloo, Waterloo,Ontario, N2L 3G1 Canada.
oNAoPH Fig. 1. Reaction catalyzedby the lanosterol 14a-demethylasefrom S.
0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)
cerevisiae.
96 Materials and Methods Compounds 1, 5, 24 and 27 were from J.T. Baker Chemicals (Phillipsburg, N J). Compounds 2, 6, 7, 8, 12, 13, 15, 25, 26, 6-methoxy-l-tetralone, 1,2,3,4-tetrahydrophenanthrene-l-one, 2-naphthaldehyde, 1-naphthylacetonitrile, 2-naphthylacetonitrile and phenanthrene-9carboxaldehyde were from Aldrich (Milwaukee, WI). 1,8-Dimethyhiaphthalene was from Fluka (Ronkonkoma, NY). Compound 23 was from Sigma (St Louis, MO). All other chemicals were of the highest quality available. S. cerevisiae JL745/pVK1 was the generous gift of Dr. J. Loper, University of Cincinnati.
Purification of S. cerevisiae P-450 P-450 was partially purified as follows. Typically, 15 1 of a 24 h culture of S. cerevisiae JL745/pVK1 grown in YEPD medium (0.5% yeast extract, 0.5% tryptone, 0.5% potassium phosphate, 3% glucose, pH 5.5) was harvested in a Sharpies continuous flow centrifuge. Cells were washed with 1.0 M sorbitol and disrupted by glass bead homogenization (Bead Beater, Biospec Prod., Bartsville, OK) for a total of 5 min (cooled in NaCl-ice slurry) (30 s disruption at 30 s cooling intervals). Cell debris was removed by centrifugation (3000 × g for 10 min) and poly(ethylene glycol) (8000 MW) was added to give a final concentration of 7.5% (w/v). Microsomes were isolated by centrifugation at 30 000 x g for 25 min. P-450 was extracted from the microsomes as described [9]. The solubilized material was applied to an octylamino Sepharose 4B column (100 ml) [101 equilibrated with 100 mM phosphate, 1 mM EDTA, 0.5~ sodium cholate and 20% (v/v) glycerol (pH 7.0) at 4 ° C. The column was washed with 2 vols. of equilibration buffer and the P-450 eluted with 100 mM phosphate, 1 mM EDTA, 0.2% (v/v) Tergitol 15-S-12 and 20% (v/v) glycerol (pH 7.0). P-450 containing fractions were pooled and concentrated over an Amicon PM30 ultrafiltration membrane to 0.5 /~M. P-450 was typically recovered in 80% final yield and with a specific content of 0.10 mnol P-450/mg protein. Spectral titrations Compounds were dissolved in ethanol (which has no detectable effect on P-450 at these concentrations) and added to a cuvette containing 800/~1 of partially purified P-450, an equal amount of ethanol was added to the reference cuvette also containing enzyme. The difference spectrum was recorded from 350-500 rim. The absorbance difference between 426 and 408 was appropriately plotted versus compound concentration using the Enzfitter (Elsevier) software to evaluate binding constants (Scatchard analysis, one binding site).
Synthesis of compounds Unless specified, all amines were taken up in diethyl ether and precipitated as their HC1 salts by passage of dry HC1 gas for 30 min for ease in purification. Physical data reported are for the isolated hydrochlorides. 1HNMR spectra were obtained on a Bruker AC200 (200 MHz) or AM250 (250 MHz) instrument. Mass spectra were recorded using chemical ionization (NH 3 as carrier gas), on a VG ZAB-E instrument by Dr. R.W. Smith (McMaster University, Hamilton, Ontario). Compound 3 was obtained in 6% yield by reductive amination of 6-methoxy-l-tetralone with sodium cyanoborohydride in the presence of ammonium acetate [11]. mp: 253-270°C dec. 1H-NMR (free base in CDC13): ~ 7.31 (d, 1H), 6.75 (dd, 1H), 6.60 (d, 1H), 3.95 (t, 1H), 3.79 (s, 3H), 2.75 (m, 2H), 1.95 (m, 2H), 1.70 (m, 2H), 1.55 (s, 2H, exchanges with D20 ). MS: m / z (relative intensity), 177 (M, 1.7%, 176 (5%), 161 (100%). Compound 20 was obtained similarly from 1,2,3,4-tetrahydrophenanthrene-l-one in 23% yield, mp: > 255°C. "H-NMR (free base in CDC13): 8 8.0 (d, 1H), 7.78 (d, 1H), 7.67 (d, 1H), 7.48 (m, 3H), 4.10 (t, 1H), 3.13 (m, 2H), 2.0 (m, 2H), 1.8 (m, 2H), 1.59 (s, 2H, exchanges with D20 ). MS: m / z (relative intensity), 198 ( M + 1, 2.2%), 181 (100%). 6-Methoxy-l-tetralone was homologated to the corresponding 1-carboxaldehyde by treatment with trimethvisulfoxonium iodide and sodium hydride as previously described [12]. Compound 4 was obtained in 6% overall yield by conversion of the aldehyde to the oxime with hydroxylamine hydrochloride in pyridine/ethanol followed by reduction with sodium borohydride in the presence of TiC14 [13] mp: 195-207°C dec. 1H-NMR (free base in CDC13): 8 7.12 (d, 1H), 6.72 (dd, 1H), 6.61 (d, 1H), 3.78 (s, 3H), 2.93 (m, 2H), 2.7 (m, 3H), 1.80 (m, 4H), 1.39 (s, 2H, exchanges with D20 ). MS: m / z (relative intensity), 192 ( M + 1, 100%), 175 (31%), 161 (38%). Similarly, compound 21 was obtained in 9% overall yield from 1,2,3,4-tetrahydrophenanthrene-l-one. rap: > 2 6 0 ° C begins to decompose at 254°C. 1H-NMR (d6DMSO): 6 8.1 (br s, 2H, exchanges with D20 ), 8.03 (d, 1n), 7.89 (d, 1n), 7.67 (d, 1H), 7.5 (m, 2H), 7.46 (d, 1H), 3.18 (m, 3H), 3.0 (m, 2H), 1.89 (m, 4H). MS: m / z (relative intensity), 212 ( M + 1, 100%), 195 (7.4%), 182 (13.4%). Compound 9 was prepared from the corresponding nitrile by reduction with sodium borohydride in the presence of CoC12 [14]. mp: 184-188°C. aH-NMR (d6DMSO): 8 8.25 (br s, 2H, exchanges with D20 ), 8.16 (d, 1n), 7.96 (d, 1H), 7.87 (d, 1U), 7.56 (m, 2I-I), 7.48 (m, 2H), 3.43 (m, 2H, becomes a triplet upon addition of D20 ), 3.11 (m, 2H). Compound 11 was similarly obtained in 25% yield from the appropriate nitrile, nap: 254-257 °C decomposes to a brown semi-solid. 1H-NMR (d6DMSO): 8 8.28
97 (br s, 2H, exchanges with D20 ), 7.88 (m, 3H), 7.76 (s, 1H), 7.50 (m, 3H), 3.15 (s, 4H, becomes a multiplet upon addition of 1)20). Compound 10 was prepared from the corresponding oxime (prepared from the commercially available aldehyde was described above) by reduction with LiA1H 4 in THF in 59% yield, mp: > 260°C begins to decompose at 245 ° C. 1H-NMR (d6DMSO): 8 8.73 (br s, 2H, exchanges with D20), 8.04 (s, 1H), 7.92 (m, 3H), 7.69 (dd, 1H), 7.55 (m, 2H), 4.20 (s, 2H). Compound 14 was obtained by bromination of 1,8dimethylnaphthalene with N-bromosuccinimide followed by treatment with 2,2,2-trifluoroacetamide and Nail in DMF to give the protected cyclic amine as previously described [15]. Hydrolysis of the amide with sodium carbonate in aqueous methanol followed by precipitation of the hydrochloride with HC1 in diethyl ether gave 14 in a 23% overall yield, mp: > 260°C, begins to brown at 170°C. aH-NMR (d6DMSO): 8 9.9 (br s, 1H, exchanges with D20 ), 7.95 (d, 2H), 7.55 (m, 4H), 4.66 (s, 4H). MS: m / z (relative intensity), 170 ( M + 1, 100%), 155 (6%). Reduction of the oxime obtained from phenanthrene-9- carboxaldehyde with NaBH 4 in the presence of TiC14 gave 16 in a 48% overall yield, mp: 246-252°C (decomposes), 1H-NMR (d 6 DMSO): 8 8.70 (m, 2H), 8.50 (br s, 2H, exchanges with D20), 7.99 (m, 1H), 7.82 (m, 2H), 7.5 (m, 4H), 4.4 (d, 2H, becomes a singlet upon addition of D 2 0 ). M S : m / z (relative intensity), 208 ( M + 1, 64%), 191 (100%). Reduction of phenanthrene-9-carboxaldehyde with sodium borohydride in methanol afforded the alcohol in a > 95% yield. Treatment with Lawson's reagent in toluene [16] gave 18 in 19% yield, mp: 82-84°C. 1HNMR (CDC13): 8 8.72 (m, 2H), 8.25 (m, 1H), 7.65 (m, 6H), 4.25 (d, 2H), 1.9 (t, 1H). MS: m / z (relative intensity), 224 (M, 39%), 191 (100%), 165 (9.7%). The alcohol could be coverted to the chloride by the method of Corey, Kim and Takedu [17] in 75% yield. Reaction with imidazole in DMF gave compound 19 in a 84% yield, mp: 118-120°C. ~H-NMR (CDC13): 8.76 (d, 1H), 8.68 (d, 1H), 7.91 (d, 1H), 7.80 (d, 1H), 7.65 (m, 5H), 7.40 (s, 1H), 7.12 (s, 1H), 6.99 (s, 1H), 5.62 (s, 2H). MS: m / z (relative intensity), 259 ( M + 1, 100%), 191 (21%), 69 (42%). Displacement of the chloride with sodium cyanide in DMF followed by reduction with LiA1H 4 in diethyl ether gave compound 17 in a 26% yield, mp: > 260°C, begins to decompose to a brown solid at 210 ° C. 1HNMR (d6DMSO): 8 8.89 (m, 2H), 8.20 (m, 1H), 7.98 (m, 3H, becomes 1H after addition of D20 ), 7.70 (m, 5H), 3.3 (m, 4H, becomes two triplets at 8 3.40 and 3.22 with the addition of D20 ). MS: m / z (relative intensity), 222 ( M + 1, 100%), 192 (21%). Compound 22 was prepared from 1-methylphenanthrene which was obtained by reaction of 1,2,3,4-tetra-
hydrophenanthrene-l-one with methylmagnesium bromide followed by dehydrogenation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. 1-Methylphenanthrene was brominated with N-bromosuccinimide and treated with hexamethyltetramine as described [18] to give 22 in a 16.5% overall yield, mp: > 260°C, begins to brown at 244°C., 1H-NMR (d6DMSO): 8 8.5 (m, 2H), 8.38 (br s, 2H, exchanges with D20), 8.07, (m, 3H), 7.75 (m, 4H), 3.19 (2, 2H, becomes a singlet upon addition of D20 ). MS: m / z (relative intensity), 208 (M + 1, 100%), 191 (79%).
Determination of MIC MICs were determined by the method of Gordon et al. [19] using final concentrations of test compounds of 500, 250, 100, 75, 50, 25, 10, 5 and 1 /xM. Tests were performed against the yeasts S. cerevisiae Y222 and S. cerevisiae 239 whose P-450 content has been previously described [20]. Results
Table I summarizes the K s or binding affinity for the various amine, imidazole and thiol liganding compounds (Fig. 2) as determined by analyses of the difference spectra generated by the yeast demethylase upon interaction with the compounds. The K s determined is a good measure of the affinity of these molecules for yeast P-450 as none of the compounds studied showed
TABLE I
Binding affinities of various probes on S. cerevisiae lanosterol demethylase Assays were conducted as outlined in Materials a n d Methods. C o m p o u n d No.
K s (#M)
C o m p o u n d No.
K s (#M)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
N.I. N.I. N.I. N.I. 331+260 112+36 a 53+9 209 5:93 505:11 580 5:423 N.I. 308 5:98 14+4
15 16 17 18 19 20 21 22 23 24 25 26 27
b
N.I., no interaction. a Some interaction, c o m p o u n d spectrum interferes. b N o interaction at 37 # M . c Strong interaction. d N o interaction at 157/~M. e N o interaction at 187 # M . r N o interaction at 276 # M .
21 + 19 N.I. c 22+2 N.I. 841 + 466 22+6 N.I. 2.6 m m s : 1.4 d e r
98
CL
NH2
~ N H
,•R
2
CH30 1
2
3, R: NH 2 4, R : CHzNH 2
{ ~NH
~ N H
2
2
6
5
7, R : NH2 8, R = CH2NH2 9, R : CH2CH2NH 2
J~ 10, 11,
R=CH2NH 2 R=CHzCH2NH 2
12, (R) 13, (S)
N H 14
]'vN HZ R
R : NH2 16, R = CH2NH;~ 17, R =CH2CH2 NH 2 18, R= CH2SH
15,
19, R=
22
20, R: NH 2 21, R =CH2NH2
CH2N,~N
0
NH2 24
23
CH3 0 . ~ H 25,
(R)
27
26, (S)
Fig. 2. Chemical structure of c o m p o u n d s utilized in this investigation.
99 which binding data could not be obtained (Fig. 4), however, this molecule appeared to show high affinity for the demethylase based on the extent of concentration-dependent spectral change. The insolubility of several of the compounds precluded a detailed study of their binding to the enzyme, however, these compounds and the highest concentrations at which little or no binding was observed are as follows: 1 (2.8 mM), 2 (1.5 mM), 3 (1.8 raM), 4 (270 #M), 12 (400 #M), 17 (80 #M), 20 (1 mM) and 23 (93 #M). In addition, a-naphthylamine 7 showed some interaction at 100 #M and 9-aminophenanthrene 15 showed no interaction with the enzyme up to 37 #M but spectral interference prevented further investigation of these amines at higher concentrations. Octanol/water partition coefficients (logP) for each compound were calculated by the method of Hansch and Leo [23]. No correlation was found between logP and K s for the compounds tested (data not shown). Minimum inhibitory concentrations for compounds 4, 10, 11, 14, 17, 18, 19 and 21 were determined against two ergosterol containing S. cerevisiae strains, one of which, Y222, contained levels of cytochrome P-450 of 36.5 pmol/mg protein and strain 239 contained levels below detection limits [20]. Strain Y222 was only susceptible to 19 (500 #M), whereas strain 239 was susceptible to compounds 10, 17 and 21 (500 #M), 14 (250 #M) and 19 (75 #M).
o~.¢l c~a0Q8C12(t 3
g
o.4o
n O0
•
|
" ~500J
I
I
I
I
5eo
I
i
I
I
I 86o
I
I
I
I
I
I
74o I 820
5.40 620 " ZOO 7.80 a Absorbance/concentrat ion
I
l
I
--
900[ 10-4 8.60 9.40
Fig. 3. Spectral titration of the demethylase with imidazole 19. The concentration of demethylase was 0.5 /~M in 100 mM potassium phosphate containing 1 mM EDTA, 0.25g Tergitol 15-S-12 and 20% glycerol (pH 7.0), 23°C; p a t h = l cm. Analysis of the titration in which the concentration of bound and free 19 were calculated using the extent of spectral change ( K s = -slope) at the wavelengths indicated. The inset shows the difference spectra obtained following additions of 4.1, 8.1, 12.1, 16.1, 20.1 and 24 #M imidazole 19.
saturation when the concentrations of ligand and enzyme were equivalent [21]. All nitrogen containing molecules gave typical Type II difference spectra [22] (peak near 426 nm and a trough at 408 nm) as exemplified by the imidazole compound 19 (Fig. 3). The 9-thiomethylphenanthrene 18 gave complex spectra from
B
I =000 !
•
360
360
400
/*40 480 Wovelength (nm)
520
560
I
•
I
•
I
•
!
•
400 440 Wcvetength (nrn)
|
.
I
.
480
600
Fig. 4. Spectral titration of the demethylase with thiol 18. The conditions of the assay were identical to that used in Fig. 3 in all respects. (A) The absolute spectra of the enzyme following additions of 0.0, 15.4, 30.5, 45.1, 59.5, 73.5 and 87.1 # M thiol 18. (B) The difference spectra obtained under the above conditions with concentrations of 18 of 7.8, 15.4, 23.0, 30.9, 37.8, 45.1 and 52.3 #M.
100 Discussion
Although problems with solubility and spectral interference with several probes were encountered in this study, which, in some cases, may have accounted for larger errors in K s determinations, general conclusions about the structure-activity relationships of these probes may still be made. Amines attached to saturated ring systems such as compounds 1-4 and 20-21 have very little affinity for the demethylase. This might be due to unfavorable conformations of the saturated ring disallowing correct positioning of the liganding amine for interaction with the iron atom. Probes having poorly basic amines, such as the aromatic amines 5-7 and 15 exhibited moderate to weak interactions with the enzyme. The spectral interference produced by several of these probes hinders a more detailed investigation with these analogues. Interposing a carbon atom between the liganding functionality and the aromatic ring systems enhanced binding to the demethylase, as shown with compounds 8 and 10. There appears to be sufficient space in the active site of the enzyme to bind both the a- and fl-substituted naphthalene probes with similar affinities. However, the enzyme becomes more discriminating against more extended amines such as the ethylamine probes 9 and 11; the a-substituted compound 9 binding much less effectively than 8 and 10 but still more effectively than 11, a result indicating a limitation to the size and/or shape of the active site for this enzyme. As there exists free rotation around one of the bonds in compound 8, the more restricted analogue 14 was prepared and studied with the enzyme. Binding affinity for the demethylase increased for this compound perhaps indicating a more restricted but favorable orientation of the amine for interaction with the iron atom. A further delineation upon the size and/or shape of the demethylase active site is the surprisingly differential interaction observed between the two methyl homologs 12 and 13 and their parent compound 8. Incorporation of an additional methyl group proximal to the amine group in 8 results in greatly reduced binding with the S isomer and little or no binding for the R isomer. Conformational analysis (unpublished observations) indicates a differential positioning of the naphthalene moieties in space for these two compounds which could then be discriminated for by the active site of the demethylase. To evaluate the effect of an additional ring structure on the binding affinity to the enzyme, various phenanthrene probes were also synthesized and studied with the demethylase. As previously mentioned, the saturated tetrahydrophenanthrenes 20 and 21 do not bind effectively with the enzyme, a result apparently confirming the importance of aromatic character to binding affinity. A similar importance of the intervening atoms between the aromatic rings and the liganding
atom can be seen in the binding affinities for compounds 15, 16 and 22 where the additional carbon atom enhances the interaction between the enzyme and the probes. The active site appears to be sufficiently large to accommodate the phenanthrenes 16 and 22 equally well and it is possible that the phenanthrene rings of these two compounds occupy identical regions in the active site. Comparing naphthalene 8 and phenanthrene 22, the additional aryl ring increases binding affinity to the enzyme and indicates that sufficient space is present to accommodate this additional ring. A similar comment could be made concerning compounds 10 and 16. The thiol containing phenanthrene, 111, would also be predicted to bind and indeed this is observed. However, comparison of the binding to the amine analogs cannot readily be made as the spectral change is fundamentally different from that produced by the amines. Thiol containing molecules have recently been utilized to probe the cytochrome P-450 enzymes from crude rat microsomes [24]. Inclusion of an additional carbon atom between the amine and the aromatic portion reduces the binding affinity as shown for compound 17. Interestingly, the imidazole, 19, binds quite effectively to the enzyme although the distance between the liganding nitrogen and the arene is much greater than that found in 16. Restricted analogues of 19 might further clarify this interaction. Various other amines were investigated as to their interaction with the demethylase. Aminoglutethimide, 23, has been shown to interact with cytochromes P-450 involved in mammalian steroid biosynthesis [25]. This compound showed no interaction with the enzyme at 93 #M. Quinoline, 24, showed low affinity for the enzyme as exemplified by its large binding constant. Quinine, guinidine and carbazole, 25, 26 and 27 were all devoid of binding affinity to the enzyme at the concentrations investigated where solubilities were not exceeded. MIC studies performed on two yeast strains containing different amounts of cytochrome P-450 indicated that several of the compounds active in our enzyme assays inhibited cell growth. Considerations such as transport and extent of protonation may be contributing factors determining the observed inhibitory potential of these compounds. In conclusion, based on the probes evaluated in this study, a discrete binding site that can accommodate planar aromatic ligands exists for this enzyme. At the very least, the enzyme can accommodate planar aromatic compounds such as naphthalene and phenanthrene as well as the sterol lanosterol. In order to increase the binding interactions between the demethylase and the non-sterol probes discussed in this study, additional interactions expected between the normal substrate, lanosterol and the demethylase should be considered. A critical hydrogen bond between the 5-keto group in camphor and tyrosine-96 in camphor hydroxylase has
101 b e e n n o t e d [5]. B a s e d o n o u r studies w i t h l a n o s t e r o l a n a l o g u e s m o d i f i e d at the 3 - h y d r o x y l p o s i t i o n ( u n p u b lished o b s e r v a t i o n s ) a n d those r e c e n t l y r e p o r t e d b y A o y a m a a n d c o - w o r k e r s [26], a s i m i l a r i n t e r a c t i o n a p p e a r s to exist b e t w e e n the 3 - h y d r o x y l g r o u p o f l a n o s t e r o l a n d a n a m i n o a c i d side c h a i n in the active site. H y d r o x y l a t e d a n a l o g u e s w h i c h t a k e this p o t e n t i a l i n t e r a c t i o n i n t o a c c o u n t c o u l d well d i s p l a y e n h a n c e d b i n d i n g . A d d i t i o n a l h y d r o p h o b i c functionalities ( a r o m a t i c , alkyl) a p p r o p r i a t e l y p o s i t i o n e d in the a n a l o g u e s h o u l d also increase the b i n d i n g affinities o f these molecules.
Acknowledgements W e wish to t h a n k Dr. J o h n L o p e r for k i n d l y p r o v i d ing the yeast strain utilized in this work. W e g r a t e f u l l y a c k n o w l e d g e the financial s u p p o r t of N S E R C ( C a n a d a ) .
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7 Lai, M.H. and Kirsch, D.R. (1989) Nucleic Acids Res. 17, 804. 8 Chen, C., Kalb, V.F., Turi, T.G. and Loper, J.C. (1988) DNA 7, 617 -626. 9 Yoshida, Y. and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660. 10 Guengerich, F.P. (1982) in Principles and Methods od Toxicology (Hayes, A.W., ed.), pp. 609-634, Raven Press, New York. 11 Borch, R.F., Bernstein, M.D. and Durst, H.D. (1971) J. Amer. Chem. Soc. 93, 2897-2904. 12 Corey, E.J. and Chaykovsky, M. (1969) Org. Syn. 49, 78-80. 13 Kano, S., Tanaka, E., Sugino, S. and Hibinu, S. (1986) Synthesis, 695-697. 14 Satoh, T., Suzuki, S., Suzuki, Y., Miyaji, Y. and Imal, Z. (1969) Tetrahedron Lett. 52, 4555-4558. 15 Hawkins, J.M. and Fu, G.C. (1986) J. Org. Chem. 51, 2820-2822. 16 Nishio, T. (1989) J.C.S. Chem. Commun., 205-206. 17 Corey, E.J., Kim, C.U. and Takeda, M. (1972) Tetrahedron Lett. 42, 4339-4342. 18 Delepine, M. (1895) Bull. Soc. Chim. Fr. 13, 358. 19 Gordon, M.A., Lapa, E.W. and Passero, P.G. (1988) J. Clin. Microbiol. 26, 1874-1877. 20 Wright, C.D. and Honek, J.F. (1989) Can. J. Mierobiol. 35, 945-950. 21 Schuster, I. (1985) Xenobiotica 15, 529-546. 22 Jefcoate, C.R. (1978) Methods Enzymol. 52, 258-279. 23 Hansch, C. and Leo, A. (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology, J. Wiley & Sons, New York. 24 Slyn'ko, N.M., Leonova, I.N., Pak, E.V., Slepneva, I.A. and Weiner, L.M. (1988) Molecular Biology (USSR) 22, 800-804 (and references therein). 25 Shaw, M.A., Nicholls, P.J. and Smith, H.J. (1988) J. Steroid Biochem. 31, 137-146. 26 Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1989) Biochina. Biophys. Acta 1006, 209-213.
