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MK386: a potent, selective inhibitor of the human type 1 5α-reductase
J. SteroidBiochem.Molec.BioLVol. 58, No. 4, pp. 377-384, 1996 Copyright© 1996 ElsevierScienceLtd. All rights reserved Printed in GreatBritain PII: $0960-0760(96)00050-7 0960-0760/96 $15.00+ 0.00
Pergamon
MK386: a P o t e n t , Selective Inhibitor o f the Human Type 1 5 -reductase K. Ellsworth, B. Azzolina, W. Baginsky, H. Bull, B. Chang, G. Cimis, S. Mitra, J. Toney, R. K. Bakshi, G. R. Rasmusson, R. L. Tolman and G. S. Harris* Departments of Enzymotogy and Medicinal Chemistry, Merck Research Laborawries, P.O. Box 2000, R80Y-140 Rahway N J 07065, U.S.A.
Steroid 5~-reductase is required for the c o n v e r s i o n o f testosterone to dihydrotestosterone. Loca!!zation o f type 1 5~-reductase in the s e b a c e o u s gland o f skin offers the possibility for selective i n h i b i t i o n o f this i s o z y m e as a t r e a t m e n t for ache. The goals o f these studies are to d e m o n s t r a t e the m e c h a n i s m o f i n h i b i t i o n o f MK386 a n d its selectivity for type 1 5~-reductase. The apparent p o t e n c y o f MK386 differed d e p e n d i n g o n the source o f the e n z y m e (i.e. r e c o m b i n a n t vs. native), yet selectivity for type 1 5~-reductase was u n c h a n g e d . O u r results indicate that the apparent p o t e n c y o f MK386 is m o d u l a t e d by the m e m b r a n e c o n c e n t r a t i o n o f the assay. T h e s e results suggest that MIO86 has a h i g h affinity for the lipid-rich m e m b r a n e e n v i r o n m e n t o f 5~-reductase. MK386 was also f o u n d to be a slow b i n d i n g inhibitor o f type 1 5~-reductase. However, the cause o f this t i m e - d e p e n d e n t i n h i b i t i o n is unrelated to partitioning o f the inhibitor into the m e m b r a n e b e c a u s e similar studies with type 2 5~-reductase indicate that MK386 is a reversible, c o m p e t i t i v e inhibitor. A n u m b e r o f c o u n t e r s c r e e n s were developed to d e m o n s t r a t e that MK386 is a p o o r inhibitor o f other steroid m e t a b o l i z i n g e n z y m e s . Copyright © 1996 Elsevier S c i e n c e Ltd.
J. Steroid Biochem. Mole,:. Biol., Vol. 58, No. 4, pp. 377-384, 1996
INTRODUCTION
inhibition of the type 1 enzyme for acne or hirsutism and inhibition of type 2 5~R for B P H or prostate cancer. T h e local conversion of testosterone (T) to the more It is now quite clear that selective inhibitors can be potent androgen 5~-dihydrotestosterone ( D H T ) by designed for type 1 and 2 5erRs. Potent, selective 5ct-reductase (5~tR) is implicated in various disease inhibitors of type 1 5~R have been reported, but no states, including benign prostatic hyperplasia (BPH), specificity data are available [6-9]. Finasteride and androgenetic alopecia, hirsutism and acne [1-3]. T h e epristeride (Fig. 1), type 2 5ctR selective inhibitors, have association of D H T with these disorders has generated undergone clinical testing for the treatment of great interest in 5ctR as a therapeutic target. However, B P H [10, 11]. Clinical studies with finasteride revealed recent identification of isozymes of 5ct R has complicated that serum levels of D H T are reduced by as m u c h as the search for clinically( useful inhibitors. T h e s e two 70%, whereas prostatic D H T decreased _> 90% at isozymes, 5~R type 1 and type 2, are differentially doses of 1 and 5 m g [12]. N o further decrease in expressed in tissues [4]. T h e type 1 enzyme is localized circulating D H T was observed at higher doses. T h e in scalp skin and liver whereas the type 2 enzyme is source of the residual circulating D H T in patients present in prostate, epididyrnis, seminal vesicles and treated with this agent is p r e s u m e d to be due to type 1 liver [4, 5]. T h e distinct tissue distribution of the 5~R, the finasteride-resistant form of the enzyme. In isozymes of 5ctR offers the possibility of selective support of this suggestion, recent studies indicate that maximal doses of M K 3 8 6 suppress serum D H T *Correspondence to: Dr G. S Harris. Tel.: + (908)594-7641; Fax: approximately 40% from baseline [9]. +(908) 594-5468. T h e high levels of type 1 5~R in scalp and other Abbreviations: T, testosterone, DHT, dihydrotestosterone, 5ctR, regions of skin as well as localization in sebaceous 5ct-reductase. Received 15 Nov. 1995; accepted 26 Jan. 1996. glands [13] are consistent with a proposal that the type 377
378
K. Ellsworth et aL
1 enzyme is important in controlling the function of sebaceous glands. In this regard, a type 1 5~R inhibitor may offer potential for the treatment of androgenetic alopecia or acne. M K 3 8 6 (Fig. 1) was previously selected to evaluate the clinical utility of an inhibitor of type 1 5~R [8]. In this study we describe the kinetic properties of M K 3 8 6 and provide evidence for its selectivity for type 1 5~R.
MATERIALS
5~R assay
T h e assays of the native and recombinant h u m a n 5~Rs were performed as described previously [5, 8]. T h e baculovirus-expressed 5ctR was provided by K. Chan and D r S. Andersson [15]. Native sources of the human type 1 and 2 5~-reductases were human scalp and prostate, respectively. Kinetic analysis
Data from progress curves of product formation were fitted to the integrated first order equation (1) where y = product, vo is the initial velocity, v~ is the velocity at infinity time and k is the pseudo first order rate constant [16]. T h e second order rate constants ( k 3 / I ~ were estimated using equation (2) where k4 (the reverse H i
Finasteride
y = Vst = (Vo - v~) (I - e-k~)/k
(1)
k = k4 + k3(I/K(1 + S/Km + I/K~))
(2)
Vo= VmaxS/(Km(1 + I/KO + s).
(3)
AND METHODS
[7-3H]Testosterone, [14C]-acetate, [7-3H]-pregneno lone, [7-3H]cholesterol, [7-3H]-androstenedione and [ 1,2,4,5,6,7,16,17-3H]-dihydrotestosterone were purchased from New England Nuclear (Boston, MA). H u m a n tissues were obtained from the International Institute for the Advancement of Medicine (IIAM, Exton, PA). M K 3 8 6 (4,7-dimethyl-4-azacholestan-3one), L-733,690 (4,7-dimethyl-4-azacholestan-5-ene3-one), dihydrofinasteride, L-651,580, L-592,887 (6fl-hydroxy-20-spirox-4-ene-3-one) and L-603,075 (17fl-ureidoandros-4-en-3-one) were provided by Drs Raman Bakshi and Gary Rasmusson. All chemicals were obtained from Sigma Chemical Co. (St Louis, MO). Protein concentrations were estimated using the method developed by Lowry [14].
= H
rate constant) was taken to be zero because the inhibition curves plateau indicating the inhibition is pseudo-irreversible under these conditions. T h e K, for formation of the preliminary EI complex was determined using equation (3)
P a t h w a y assay for conversion o f acetate to cholesterol
T h e effects of M K 3 8 6 on the h u m a n hepatoma cell-line H e p G 2 were examined. H e p G 2 cells were plated in 6-well plates and grown overnight in D M E M supplemented with 10% serum. Cells were grown for an additional 24 h in the presence of 2 ml medium supplemented with 10% delipidated serum to maximize cholesterol biosynthesis. T h e cells were treated with M K 3 8 6 at various concentrations for 20 h. After 18 h, 5 mCi of 14C-acetate was added to each well. T h e cells were then extracted with 1 ml 1 N N a O H , saponified by heating at 95°C for 1 h, neutralized with HCI and extracted with 3 x 5 ml petroleum ether. T h e extracts were evaporated to dryness under a stream of nitrogen, resuspended in 1 0 0 / d ethyl acetate and applied to PE SIL G plates (Whatman, Clifton, N'J). Cholesterol was separated from other non-saponifiable lipid components by thin layer chromatography on silica gel plates using petroleum ether:diethyl ether:glacial acetic acid (75:25:1). T h e incorporation of 14C into cholesterol and squalene was quantified using the phosphorimager and Imagequant application software. A s s a y for the conversion of cholesterol to pregnenolone
H u m a n adrenal tissue was pulverized with a freezer mill and resuspended in three volumes of 25 m M Tris, p H 7.4, 1 m M E D T A , 250 m M sucrose and 20% glycerol. T h e suspension was homogenized with a Dounce homogenizer and the crude homogenate used as the source of enzyme in this assay. T h e reaction mixture contained 35 m M sodium phosphate p H 7.4, 20 m M potassium chloride, 5 m M magnesium chlor-
H
HOOC
! CH3 Epristeride
Fig. 1. Inhlbitors o f 5~-reductase.
MK386
MK386: A Potent, Selective Inhibitor of the Human Type 1 5~-Reductase ide, 0.2 m M
E D T A , 2 0 0 m M sucrose, 6 p M [710 m M isocitrate, and 1 m M N A D P H in a final volume of 50 pl. M K 3 8 6 was added to the assay mixture to give a tinal concentration of 1000 n M in 0.5% ethanol. L-592,887 (6fl-hydroxy-20-spirox-4ene-3-one), an inhibitor of the side chain cleavage enzyme, was included a:~ a positive control in the assay at a concentration of 50 pM. T h e reaction was initiated by the addition of adrenal homogenate and incubated for 2 h at 37°C. T h e mixture was quenched with cyclohexane:ethyl acetal:e (36:64 v/v) and the aqueous and organic layers were separated by centrifugation at 14,000 rpm in an E p p e n d o r f microfuge. T h e organic layer was subjected to normal phase H P L C (25 cm Whatman partisil 5 silica column equilibrated in 1.2 ml/min cyclohexane:ethyl acetate (36:64 v/v), retention times cholesterol, 5.4 min; pregnenolone, 8 min).
3H]cholesterol,
Pathway assay for the conversion of pregnenolone to 5-androstenediol H u m a n testicular tissue was pulverized in liquid nitrogen using a freezer :mill and homogenized in 1 m M M O P S p H 7.2, 100 m M potassium chloride, 1 m M E D T A , 2 m M D T T containing 1 M sucrose using a Potter-Elvehjem homogenizer. This crude homogenate was used as the source of enzymes in this assay. T h e reaction mixture contained 40 m M potassium phosphate, p H 7.5, 0.09 # M [7-3H]-pregnenolone, 1 m M D T T , 0.5 p M N A D P + and 3.6 p M NAD÷in a final volume of 100 pl. M K 3 8 6 was added to the assay mixture to give a final concentration of 1 n M - 1 0 # M inhibitor in 0.5% ethanol. L-603,075 (17fl-ureidoandros-4-en-3-one), an inhibitor of the C17,20lyase, was added as a positive control in this assay. T h e reaction was initiated by the addition of testicular homogenate corresponding to 3 mg ]protein and incubated at 37°C. After 10 min the reaction was quenched by extraction with 290 pl of cyclohexane:ethyl acetate (70:30 v/v) containing 10 #g each unlabelled dehydroepiandrosterone (DHEA), androstenedione and testosterone. T h e organic layer was subjected to normal phase H P L C (25 cm W h a t m a n partisil 5 silica column equilibrated in 1.2 ml/min cyclohexane:ethyl acetate (80:20 v/v), retention times pregnenolone, 12 min; 17ct-hydroxypregnenolone, 7.5 min; dehydroepiandrosterone, 15 min; 5-androstenediol, 20 min). T h e conversion of pregnenolone to 17~-hydtoxypregnenolone by 17~-hydroxylase, subsequent conversion to D H E A by 17=~-hydroxypregnene (;1720 lyase and further conversion to 5-androstenediol by 17fl-hydroxysteroid dehydrogenase was monitored using the radioactivity flow detector. Following a 10 rain incubation under the assay conditions described, the product distribution was 50% pregnenolone, 5% 17~-hydroxypregnenolone, 10% dehydroepiandrosterone and 23% 5-androstenediol.
379
Aromatase assay A sample of h u m a n placenta was homogenized in two volumes 1 m M M O P S , p H 7.2, 100 m M potassium chloride, 1 m M E D T A , 2 m M D T T and 1 M sucrose using a Brinkmann Instruments Polytron followed by standard D o u n c e homogenization (15 x). T h e sample was centrifuged at 1 0 , 0 0 0 x g for 10 min, the supernatant was collected and centrifuged at 100,000 x g for 1 h at 4°C. T h e microsomal pellet was washed once and resuspended in buffer and stored frozen at -80°C. T h e reaction mixture for the aromatase assay contained 50 m M potassium phosphate, p H 7.5, 500 # M N A D P H , 0.5 m M E D T A , 0.5 m M D T T , 40 n M [7-3H]-androstenedione, 5 m M magnesium chloride and 3.1 pg h u m a n placental microsomes in a final volume of 100 pl. T h e assay was initiated by the addition of enzyme and incubated at 37°C. M K 3 8 6 was added to the assay mixture to give a final concentration of 1 n M - 1 0 # M in 0.5% ethanol. A T D (1,4,6-androstatriene-3,17-dione), a known inhibitor of aromatase was included as a positive control in the assay. After 20 min the reaction was quenched by extraction with 250#1 of a mixture of cyclohexane:ethyl acetate (64:36v/v) containing 1 0 p g each T , D H T and androstenedione. T h e organic layer was subjected to normal phase H P L C (25 cm Advantage Plus 5 micron silica column equilibrated in 1.2 ml/min cyclohexane: ethyl acetate (70:30v/v); retention times estrone, 5.0 min; estradiol, 7.5 min; androstenedione, 11 min. T h e conversion of androstenedione to estrone was monitored using the radioactivity flow detector by mixing the H P L C effluent with two volumes of Flo Scint II (Radiomatic, T a m p a , FL).
17fl-hydroxysteroid dehydrogenase Samples of liver were pulverized using a freezer mill and homogenized in 40 m M potassium phosphate, p H 6.5, 5 m M magnesium sulphate, 25 m M potassium chloride, 1 m M phenylmethylsulfonyl fluoride, 1 m M D T T containing 0.25 M sucrose using a Potter-Elvehjem homogenizer. T h e sample was centrifuged at 11,500 x g for 15 min. T h e supernatant from this step was collected and subjected to a final centrifugation at 105,000 x g for 1 h at 4°C. T h e microsomal pellet was washed two times and resuspended in two volumes of buffer. Glycerol was added to the resuspended pellet to a final concentration of 20%. T h e reaction mixture contained 40 m M potassium phosphate, p H 7.5, 2 m M N A D ÷, 1 m M E D T A , 5 m M D T T and 2 # M [7-3H]-T in a final volume of 100 pl. M K 3 8 6 was added to the assay mixture to give a final concentration of 1 n M - 1 0 # M in 0.5% ethanol. T h e assay was initiated by the addition of 0.4 #g human liver microsomes and incubated at 37°C. After 20 rain the reaction was quenched by extraction with 250 pl of a mixture of cyclohexane:ethyl acetate (64:36v/v) containing 10 #g each T, D H T and androstenedione.
380
K. Ellsworth et al.
T h e organic layer was subjected to normal phase H P L C (25 cm Advantage Plus 5 micron silica column equilibrated in 1.2 ml/min cyclohexane: ethyl acetate (64:36 v/v); retention times androstenedione, 8.5 min; testosterone, 11.5 min). T h e conversion of T to androstenedione was monitored using the radioactivity flow detector by mixing the H P L C effluent with two volumes of Flo Scint II (Radiomatic).
Table 1. Inhibition of 5ct-reductase
ICs0 (nM) Inhibitor MK386 MK386 Dihydrofinasteride Dihydrofinasteride L-651,580 L-651,580
Enzyme source
Type 1
Type 2
Native Recombinant Native Recombinant Native Recombinant
20 0.9 700 540 40 43
3100 150 15 6 960 720
H u m a n androgen receptor assay
T h e stable transfectant cell line expressing the h u m a n androgen receptor (hAR) was provided by Prof. Michael McPhaul at the Southwestern Medical School in Dallas, T X [17]. T h e cell line was maintained in H a m ' s F12 m e d i u m (Gibco, G r a n d Island, NY) supplemented with 10% fetal calf serum (Gibco), P S N antibiotic mixture (Gibco) and 0 . 4 m g / m l G418 (Gibco). T h e cells are fed three times weekly, and are maintained at 37°C with 5% CO2. Cells are grown to near confluence, and are washed twice with PBS (phosphate buffered saline). T h e cells are harvested and the pellet is washed twice with T E G M ( 1 0 m M Tris-HC1; 1 m M E D T A ; 10% glycerol; 1 m M beta-mercaptoethanol, 10 m M sodium molybdate; p H 7.2) buffer. T h e cell suspension was frozen in liquid nitrogen and stored at -80°C. Prior to the assay, the suspension was thawed at 4°C and centrifuged for 1 h at 45,000 r p m (Ti70) at 4°C to remove cellular debris. [1,2,4,5,6,7,16,17-3H]DHT was added to the extract (supematant) to give a final concentration of 1 nM. Typically the assays were done in triplicate by dispersing 0.1 ml of the s u p e m a t a n t into borosilicate glass tubes containing inhibitor. T h e final concentration (v/v) of ethanol does not exceed 1% in the assay. T h e mixture was incubated overnight at 4°C. Free [ 3 H ] D H T was separated from receptor-bound h o r m o n e by the addition of 0.1 ml of dextran coated charcoal (0.5% dextran T - 7 0 , 0.1% gelatin; 5% charcoal in 10 m M Tris, p H 7.0, 0.1 m M E D T A ) . T h e suspension was centrifuged for 15 min at 5000 rpm. T h e 3H in the supernatant was determined by scintillation counting and represented the receptorbound DHT.
RESULTS
Inhibition o f the h u m a n type 1 and 2 5c~R
Initial studies to identify a selective inhibitor of 5~R were carried out using h u m a n scalp and prostate as sources of the h u m a n type 1 and 2 5c~Rs, respectively. T h e results of these analyses led to the discovery of M K 3 8 6 , a 4-N-methyl azasteroid [8]. As indicated in Table 1, M K 3 8 6 is a potent inhibitor of type 1 5c¢R in h u m a n scalp skin with an IC50 of ,,~ 20 nM. This c o m p o u n d is a poor inhibitor of type 2 5~R in h u m a n prostate with an IC~0 of ~ 3100 nM. These results indicate that the selectivity is > 150-fold for type 1
5~R. Similar selectivity is found using recombinant type 1 and 2 expressed in baculovirus, although the ICs0 values for M K 3 8 6 are ~ 20-fold lower than those obtained using the native enzymes (Table 1). Interestingly, no differences are noted in ICs0 values for dihydrofinasteride or L-651,580 which are two standards used in the assays to compare native and recombinant 5ctR. T h e difference in IC~0 values for M K 3 8 6 with native and recombinant 5~Rs was unexpected, and appeared to be restricted to the aza-cholestan-3-one class of inhibitors. Additional experiments were designed to explore the cause of the apparent potency differences with MK386. One obvious feature of this c o m p o u n d to consider is the hydrophobicity. It is reasonable to assume that M K 3 8 6 preferentially binds to protein or partitions into the m e m b r a n e rather than behave as a freely soluble inhibitor. Any effect of protein binding on the apparent potency can be ruled out since ICs0 values for the native enzyme were unchanged in the presence of 20 mg/ml BSA or 50% fetal calf serum (data not shown). A more likely possibility is that the hydrophobicity of the c o m p o u n d leads to significant partitioning into the m e m b r a n e , ultimately leading to a decrease in the solution concentration and an increase in the m e m b r a n e concentration. In this case, the higher specific activity of the recombinant enzyme results in significantly lower amounts of m e m b r a n e in the assay mixture c o m p a r e d to the native enzyme. I f the difference in m e m b r a n e concentration in the assay mixture gives rise to the discrepancy in the IC~0 values for M K 3 8 6 with native and recombinant 5~R, then addition of control (non-5~R containing) m e m b r a n e s should alter the apparent potency of M K 3 8 6 while inhibition of the enzyme by the standard c o m p o u n d s should not be affected. In order to test the m e m b r a n e partitioning hypothesis, the ICs0 for M K 3 8 6 for the recombinant type 2 5~R was determined in the presence of variable amounts of control m e m b r a n e s prepared from uninfected baculovirus. As listed in Table 2, the ICs0 for M K 3 8 6 increased with increasing amounts of m e m b r a n e , approaching those obtained with native type 2 5~R. T h e same dependence of ICs0 value on m e m b r a n e concentration was observed with L-733,690, a related 4-aza-cholestan-3-one. As predicted, IC~0 values in control experiments with
MK386: A Potent, Selective Inhibitor of the Human Type 1 5~t-Reductase dihydrofinasteride and L-651,580 are not altered as a function of the membrane concentration. F r o m these experiments, we conc/'ade that the potency differences observed with native and recombinant 5~tR are not due to differences in the enzyme, but simply reflect changes in the membrane concentration in the assay mixture. At present it is unclear whether the enzyme is exposed to the aza-cholestan-3-ones from the membrane or the solution.
11.0 l
7.3
n
Mechanism of inhibition
3.7
As illustrated in Fig. 2A, progress curves of D H T formation by native type 1 5~R are non-linear in the presence of M K 3 8 6 and indicate that this inhibitor is more potent than predicted from a fixed time point ICs0 determination. Similar results were obtained using either native or recombinant enzyme; therefore, the time-dependent inhibition does not reflect membrane influences on potency but direct interaction with the enzyme. T h e data in Fig. 2A, obtained using native type 1 5~R, indicate this inhibitor is slow binding and forms an initial complex with a Ki= 14 n M which then rearranges to a high affinity complex with a pseudo first order rate constant of 3 x 10 -3 s-L T h e second order rate constant is kJKi = 2.5 x 105 M - ~s - 1. These data are consistent with slow-binding inhibition as described by Morrison and Walsh [16] where inhibitor binds to enzyme to produce a loosely associated Michaelis complex EI which isomerizes to give EI*, a tightly b o u n d complex (Fig. 1) which for M K 3 8 6 gives a dissociation constant Scheme <100 pM. Although a similar kinetic scheme had been described for the inhibition of 5~R by finasteride [18, 19], the mechanism of inhibition for M K 3 8 6 is different because it is unable to undergo the reduction step as found with finasteride [20]. Ki
Scheme 1
381
It3
F, + I ~.~ E:I ~ E:I* k4
In contrast to that found for native type 1 5~R, the time course for inhibition for native type 2 5~R by M K 3 8 6 indicates that this c o m p o u n d is a classic reversible inhibitor of dais enzyme (Fig. 2b). In this Table 2. Influence of membrane concentration on inhibition of Oepe 2 5M~! by MK386 IC~0 (nM) Enzyme source control microsomes
r5~R 0 pg
r5~R 0.9 p g
r5~R 45 p g
Native 5c~R
Compound MK386 L-733,690 Dihydrofinasteride L-651,580
63 100 6 630
75 240 7 780
700 5000 4 700
3100 > 1000 15 960
Assays were carried out as described in M e t h o d s with type 2 5~R produced recombinantly (r5~R) using baculovirus or from h u m a n prostate (native) as described previously [5]. Control microsomes were obtained from n o n - 5 ~ R producing baculovirus a n d were included in the 100 #1 assay as indicated.
•
:
8
24 nM I
00
J
42
84
126
min 19.0 Type 2
/ aM
12.7 -
"0
.o
6.4
00 °
I
1
42
84
126
min
Fig. 2. I n h i b i t i o n o f 5 ~ - r e d u c t a s e b y M K 3 8 6 . A , R e a c t i o n p r o g r e s s c u r v e s for n a t i v e t y p e 1 5 ~ - r e d u c t a s e i n a s s a y m i x t u r e c o n t a i n i n g 0.1 M p o t a s s i u m p h o s p h a t e , p H 7.2, 5 / i M [7-3H]T , 5 0 0 / ~ M N A D P H w i t h M K 3 8 6 as i n d i c a t e d . B , R e a c t i o n p r o g r e s s for n a t i v e t y p e 2 5 ~ - r e d u c t a s e in an a s s a y m i x t u r e c o n t a i n i n g 40 m M s o d i u m citrates p H 5.5, 0 . 3 / * M [ 7 - ' H ] - T , 5 0 0 / ~ M N A D P H a n d M K 3 8 6 as i n d i c a t e d .
case, additional mechanistic studies are more straightforward and are not complicated by the time-dependent nature of the inhibition as found with type 1 5ctR. In order to minimize the effective dilution of inhibitor and consequently obtain the best estimate of the intrinsic potency of the c o m p o u n d , recombinant 5~t-reductase type 2 was used as the source of enzyme in these studies. As indicated in Fig. 3, M K 3 8 6 exhibits competitive inhibition of type 2 5~tR vs. T with an apparent / ~ = 9 0 nM. Therefore, despite the influence of the membrane concentration on the apparent potency, the inhibition of 5~R by M K 3 8 6 is not a consequence of altering the membrane environment of the enzyme, but is due to binding at the enzyme active site.
382
K. Ellsworth et aL 5.7
4.6
2.3 3.4
g
1.1
I
3.0
I
I
6.0 9.0 I/[Testosterone] (gM -1)
I
12.0
Fig. 3. C o m p e t i t i v e i n h i b i t i o n o f t h e r e c o m b i n a n t t y p e 2 5 ~ - r e d u c t a s e b y M K 3 8 6 . T h e e n z y m e w a s i n c u b a t e d in a m i x t u r e c o n t a i n i n g 40 m M s o d i u m c i t r a t e , p H 5.5, 5 0 0 / i M N A D P H , 0.3/~M [7-3H]-T a n d M I O 8 6 a s i n d i c a t e d .
Selectivity of M K 3 8 6
Lack of effect on the conversion of cholesterol to pregnenolone
Previous work in the azasteroid series demonstrated that additional activities were associated with some of the 4-N-methyl analogs. Most notably, 4 M A (17flN,N-diethylcarbamoyl-4-methyl-4-aza-5~-androstan3-one) was found to interact with the rat androgen receptor [21], as well as to inhibit the bovine adrenal 3fl-hydroxy-5-ene-steroid dehydrogenase/3-keto-5-enesteroid isomerase [22]. Given the structural similarity of the A ring of M K 3 8 6 to 4MA, it was prudent to demonstrate the specificity of M K 3 8 6 for type 1 5~R. A n u m b e r of counterscreens were developed to test the selectivity of this c o m p o u n d with the h u m a n enzymes. Several pathway assays were developed to monitor potential effects of M K 3 8 6 on enzymes involved in the overall conversion of acetate to cholesterol, as well as further conversion to testosterone or estrogens. These pathway assays facilitate the survey of multiple enzymes in a single mixture. Inhibition of any of these steps is unacceptable in a candidate slated for clinical development because of potential effects on the formation of testosterone, glucocorticoids, mineralocorticoids or estrogens.
T h e conversion of cholesterol to pregnenolone requires the action of the C20-hydroxylase, C22-hydroxylase and the C20,22 side chain cleavage enzyme. Although M K 3 8 6 is a 4-azasteroid, the cholesterol side chain at C,7 could permit this c o m p o u n d to act as an alternate substrate or inhibitor of these enzymes and potentially block the initial steps in the biosynthesis of all steroid hormones. In order to determine whether M K 3 8 6 is an inhibitor of these enzymes, an assay was developed to monitor the conversion of radiolabelled cholesterol to pregnenolone using h u m a n adrenal homogenate, a major site of steroid h o r m o n e biosynthesis. T h e results of these studies indicate that at 10,000 n M M K 3 8 6 there is no effect on the production of pregnenolone (Table 3). Lack of effect on the conversion of pregnenolone 5-androstenediol
to
As indicated above, M K 3 8 6 is a 4-azasteroid which could potentially inhibit other steroid metabolizing enzymes besides 5~R. In order to determine whether this c o m p o u n d inhibits 17~-hydroxylase, 17a-hydroxyTable 3. Selectivity of MK386 for type 1 5c~-reductase
Lack of effect on the conversion of acetate to cholesterol
Enzyme/pathway
ICso (nM)
An assay in H E P G 2 cells was used to monitor the overall conversion of acetate to squalene or cholesterol. Cells were grown in the presence and absence of M K 3 8 6 , in the presence of delipidated serum to upregulate cholesterol biosynthesis and then supplemented with [14C]acetate. T h e incorporation of 14C into squalene and cholesterol was monitored. M K 3 8 6 is a p o o r inhibitor of squalene and cholesterol biosynthesis with ICs0 values of 5 0 0 0 n M and 10,000 nM, respectively (Table 3).
5g-reductase type 1' 5g-reductase type 2 a Acetate ~ squalene Acetate ~ cholesterol Cholesterol ~ pregnenolone Pregnenolone --, 5-androstenediol 17fl-hydroxysteroid dehydrogenase type 2 Aromatase 3fl-hydroxysteroid dehydrogenase/isomerase Androgen receptor
< 20 3100 5000 10,000 > 1000 > 10,000 > 10,000 > 10,000 8000 > 100,000
aValues obtained using native sources of 5~-reductase.
MK386: A Potent, Selective Inhibitor of the Human Type 1 5ct-Reductase p r e g n e n e C17,20-1yase o r t y p e 3 1 7 f l - h y d r o x y s t e r o i d dehydrogenase, a modification of the system developed b y Y a n a i h a r a a n d T r o e n [23] was u s e d to m o n i t o r t h e c o n v e r s i o n o f r a d i o l a b e l l e d p r e g n e n o l o n e to 5 - a n d r o s t e n e d i o l . H u m a n t e s t i c u l a r tissue, a m a j o r site o f a n d r o g e n b i o s y n t h e s i s , was u s e d as a s o u r c e o f t h e s e e n z y m e s for these assays. M K 3 8 6 h a d n o effect o n the c o n v e r s i o n o f p r e g n e n o l o n e to 5 - a n d r o s t e n e d i o l w h e n t e s t e d u p to 10,000 n M ( T a b l e 3). T h e r e f o r e , M K 3 8 6 does not inhibit the human testicular 17~-hydroxylase, 17~-hydroxypregnene (217_20 lyase or 1 7 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e . I n c o n t r a s t , L - 6 0 3 , 0 7 5 (17flureidoandros-4-en-3-one) a compound previously r e p o r t e d as an i n h i b i t o r o f t h e C17,20-1yase [24] a p p e a r e d to b l o c k t h e 1 7 ~ - h y d r o x y l a s e a n d r e s u l t e d in 8 4 % i n h i b i t i o n o f the p a t h w a y at 10,000 n M . L a c k of inhibition o f 3fl-hydroxysteroid dehydrogenase/isomerase P r e v i o u s studies b y B r a n d t a n d L e v y [22] i n d i c a t e t h a t several 4 - N - m e t h y l a z a s t e r o i d s are p o t e n t i n h i b i tors o f t h e b o v i n e 3 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e / i s o m e r a s e w i t h low n M K, values. T o date, little w o r k has b e e n d o n e to s t u d y t h e i n h i b i t i o n o r s t r u c t u r e - a c tivity r e l a t i o n s h i p o f t:he h u m a n 3 f l - h y d r o x y s t e r o i d dehydrogenase/isomerase by 4-azasteroids. These studies w e r e i n i t i a t e d to d e t e l a n i n e the affinity o f M K 3 8 6 for human type 1 3fl-hydroxysteroid dehydrogenase/isom e r a s e f r o m h u m a n p l a c e n t a , a n essential e n z y m e in t h e f o r m a t i o n o f m a n y o f the s t e r o i d h o r m o n e s . M K 3 8 6 is a p o o r i n h i b i t o r o f t y p e 1 3 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e / i s o m e r a s e a n d is n o t e x p e c t e d to effect the production of glucocorticoids, mineralocorticoids, p r o g e s t i n s o r a n d r o g e n s ( T a b l e 3). L a c k o f inhibition o f 17fl-hydroxysteroid dehydrogenase T h e i n t e r c o n v e r s i o n o f a n d r o s t e n e d i o n e to t e s t o s t e r o n e a n d e s t r o n e to e s t r a d i o l is c a t a l y s e d b y 17fl-hyd r o x y s t e r o i d d e h y d r o g e n a s e . M u l t i p l e i s o z y m e s o f this e n z y m e exist w h i c h m a y differ in sensitivity to M K 3 8 6 . Results obtained using human testicular homogenates to m o n i t o r the c o n v e r s i o n o f p r e g n e n o l o n e to 5 - a n d r o s t e n e d i o l i n d i c a t e t h a t t h e r e is n o i n h i b i t i o n o f t h e t e s t i c u l a r (type 3) 1 7 f l - h y d r o x y s t e r o i d d e h y d r o g e n ase. A s e c o n d assay was d e v e l o p e d to d e t e r m i n e the effects o f M K 3 8 6 o n t y p e 2 1 7 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e , t h e form f o u n d in h u m a n liver [25]. N o i n h i b i t i o n o f this isofonaa was o b s e r v e d w i t h M K 3 8 6 ( T a b l e 3). L a c k of inhibition o f aromatase T h e p o t e n t i a l use o f a t y p e 1 5c~R i n h i b i t o r for the t r e a t m e n t o f a c n e or h i r s u t i s m in w o m e n r e q u i r e s t h a t t h e r e b e n o effects o n the b i o s y n t h e s i s o f estrogen. N o i n h i b i t i o n o f h u m a n p l a c e n t a l a r o m a t a s e was o b s e r v e d in t h e p r e s e n c e o f M K 3 8 6 (ICs0 > 10,000 n M , T a b l e 3). T h e r e f o r e , this c o m p o u n d s h o u l d n o t interfere w i t h t h e p r o d u c t i o n o f e s t r o g e n !in w o m e n .
383
N o detectable binding to the h u m a n androgen receptor M o s t o f t h e studies to define the s t r u c t u r e activity r e l a t i o n s h i p for b i n d i n g o f a z a s t e r o i d s to the a n d r o g e n r e c e p t o r have f o c u s e d o n the r e c e p t o r f r o m rat r a t h e r t h a n h u m a n [26]. T h e r e f o r e , d e s p i t e t h e w e a l t h o f i n f o r m a t i o n d e s c r i b i n g t h e b i n d i n g c h a r a c t e r i s t i c s o f the r a t a n d r o g e n r e c e p t o r , little i n f o r m a t i o n was available to p r e d i c t w h e t h e r M K 3 8 6 w o u l d b i n d to t h e h u m a n a n d r o g e n r e c e p t o r . I n o r d e r to o v e r c o m e this l i m i t a t i o n , a s c r e e n i n g assay was d e v i s e d u s i n g t h e c l o n e d h u m a n a n d r o g e n r e c e p t o r [17, 27]. R e c e p t o r b i n d i n g assays o f M K 3 8 6 r e v e a l e d t h e r e is n o d e t e c t a b l e b i n d i n g to the c l o n e d h u m a n r e c e p t o r ( T a b l e 3). T h i s r e s u l t i n d i c a t e s t h e r e is > 5 0 0 0 - f o l d selectivity for t y p e 1 5 ~ R c o m p a r e d to the h u m a n a n d r o g e n r e c e p t o r .
CONCLUSION
T h e results d e s c r i b e d in this p a p e r i n d i c a t e t h a t M K 3 8 6 is a p o t e n t i n h i b i t o r for h u m a n t y p e 1 5ciR. T h e l a c k o f effect o f M K 3 8 6 in any o f the c o u n t e r s c r e e n s suggests that, w h e n u s e d clinically, M K 3 8 6 is selective for t h e t a r g e t e n z y m e , t y p e 1 5~R. T h e availability o f this i n h i b i t o r d e f i n e d t h e c o n t r i b u t i o n o f t y p e 1 5 ~ R to c i r c u l a t i n g D H T levels in h u m a n s [9]. G i v e n t h e l o c a l i z a t i o n o f t h e t y p e 1 i s o z y m e in t h e s e b a c e o u s g l a n d a n d t h e l i p i d - r i c h s e c r e t i o n o f the g l a n d , t h e h y d r o p h o b i c p r o p e r t i e s o f the i n h i b i t o r m a d e it an attractive c a n d i d a t e for the t r e a t m e n t o f acne.
REFERENCES
1. Metcalf B. W., Levy M. A. and Holt D. A.: Inhibitors of 5ct-reductase in benign prostatic hyperplasia, male pattern baldness and acne. Trends Pharmac. Sci. 10 (1989) 491-495. 2. Li X., Chem C., Singh S. M. and Labrie F.: The enzyme and inhibitors of 4-ene-3-oxosteroid 5ct-reductase. Steroids 60 (1995) 430-441. 3. Brooks J. R., Harris, G. S. and Rasmusson G. R.: Inhibitors of steroid 5ct-reductase. In Design of Enzyme Inhibiwrs as Drugs (Edited by M. Samdler and H. J. Smith). Oxford University Press, Oxford (1994) pp. 495-518. 4. Thigpen A. E., Silver R. I., Guileyardo J. M., Casey M. L., McConnell J. D. and Russell D. W.: Tissue distribution and ontogeny of steroid 5~-reductase isozyme expression. J. Clin. Invest. 92 (1993) 903-910. 5. Harris G., Azzolina B., Baginsky W., Cimis G., Rasmusson G. H., Tolman R. L., Raetz C. R. H. and Ellsworth K.: Identification and selective inhibition of an isozyme of steroid 5c~-reductase in human scalp. Proc. Nam. Acad. Sci. U.S.A. 89 (1992) 10787-10791. 6. Jones C. D., Audia J. E., Lawhorn D. E., McQuaid L A., Neubauer R. L., Pike A. J., Pennington P. A., Stamm N. B., Toomey R. E. and Hirsch K. S.: Nonsteroidal inhibitors of human type 1 5~-reductase. J. Med. Chem. 36 (1993) 421-423. 7. Hirsch K. S., Jones C. D. A. J. E., Andersson S., McQuaid L., Stature N. B., Neubauer B. I., Pennington P. and Toomey R. E.: LY191704: a selective, nonsteroidal inhibitor of human steroid 5~-reductase type 1. Proc. Nam. Acad. Sci. U.S.A. 90 (1993) 5277-5281. 8. Bakshi R. K., Patel G. F., Rasmusson G. H., Baginsky W. F., Cimis G., Ellsworth K., Chang B., Bull H., Tolman T. L. and Harris G. S.: 4,7fl-Dimethyl-4-azacholestan-3-one (MK386) and related 4-azasteroids as selective inhibitors of human type 1 5ct-reductase. J. Med. Chem. 37 (1994) 3871-3874. 9. Schwartz J., Van Hecken A., De L. I., Wang D., Depre M., De
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Schepper P. and Gertz B.: Pharmacodynamics of MK386--A novel inhibitor of 5ct-reductase. Clin. Pharmacol. Ther. 57 (1995) 171. Gormley G. J., Stoner E., Rittmaster R. S., Gregg H., Thompson D. L., Lasseter K. G., Vlasses P. H. and Stein E. A.: Effects of fmasteride (MK906), a 5~-reductase inhibitor, on circulating androgens in male volunteers, ft. Clin. Endocr. Metab. 70 (1990) 1136-1141. Audet P., Nurcombe H., Lamb Y., Jorasky D., Lloyd-Davis K. and Morris X. X.: Effect of multiple doses of epristeride (E) a steroid 5~-reductase inhibitor on serum dihydrotestosterone in older male subjects. Clin. Pharmacol. Ther. 53 (1993) 231-233. McConnell J. D., Wilson J. D., George F. W., Geller J., Pappas F. and Stoner E.: Finasteride, an inhibitor of 5~-reductase suppresses prostatic dihydrotestosterone in men with benign prostatic hyperplasia, ft. Clin. Endocr. Metab. 74 (1992) 505-508. Thiboutot D., Harris G., Iles V., Cimis G., Gilliland K. and Hagari S.: Activity of the type 1 5~-reductase exhibits regional differences in isolated sebaceous glands and whole skin. ft. Invest. Dermatol. 105 (1995) 209-214. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent, ft. Biol. Chem. 193 (1951) 265-275. Chan H. K., Geissler W. M. and Andersson S.: Characterization of human steroid 5~-reductase type 1 and 2 expressed in the baculovirus system. In Sex Hormones and Antihormones in Endocrine Dependent Pathology: Basic and Clinical Aspects (Edited by M. Motta and M. Serio). Elsevier Science M.V., Amsterdam (1994), pp. 67-76. Morrison J. F. and Walsh C. T.: The behavior and significance of slow-binding enzyme inhibitors. Adv. Enzymol. Relat. Areas Molec. Biol. 61 (1988) 201-301. Tilley W. D., Marcelli M., Wilson J. D. and McPhaul M. J.: Characterization and expression of a cDNA encoding the human androgen receptor. Proc. Nam. Acad. Sci. U.S.A. 86 (1989) 327-331. Tian G., Stuart J. D., Moss M. L., Domanico P. L., Bramson
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H. N., Patel I. R., Kadwell S. H., Overton L. K., Kost T. A., Mook Jr, R. A., Frye S. V., Batchelor K. W. and Wiseman J. S.: 17/~-(N-tert-butylcarbamoyl)-4-aza-5~-androstan-1-en-3-one is an active site directed slow time-dependent inhibitor of human steroid 5~-reductase 1. Biochem. 33 (1994) 2281-2296. Faller B., Farley D. and Nick H.: Finasteride: A slow-binding 5a-reductase inhibitor. Biochemistry 32 (1993) 5705-5710. Baginsky W. F., Harris G. and Bull H.: Mechanism of inhibition of human prostatic 5~-reductase by 4-azasteroids. F A S E B J. 8 (1994) A638. Liang T. and Heiss C. E.: Inhibition of 5a-reductase, receptor binding and nuclear uptake of androgens in the prostate by a 4-methyl-4-aza-steroid. J. Biol. Chem. 256 (1981) 7998-8005. Brandt M. and Levy M.: 3~-hydroxy-AS-steroid dehydrogenase/ 3-keto-A~-steroid isomerase from bovine adrenals: Mechanism of inhibition by 3-oxo-4-azasteroids and kinetic mechanism of the dehydrogenase. Biochemistry 28 (1989) 140-148. Yanaihara T. and Troen P.: Studies of the human testis. 1. Biosynthetic pathways for androgen formation in human testicular tissue in vitro. J. Clin. Endocr. Metab. 34 (1972) 783-792. Arth G. E., Patchett A. A., Jefopoulus T., Bugianesi R. L. and Peterson L. H.: Steroidal androgen biosynthesis inhibitors. J. Med. Chem. 14 (1971) 675-679. Casey M. L., MacDonald P. C. and Andersson S.: 17~-hydroxysteroid dehydrogenase type 2: chromosomal assignment and progestin regulation of gene expression in human endometrium. J. Clin. Invest. 94 (1994) 2135-2141. Rasmusson G., Reynolds G., Steinberg N., Walton E., Patel G., Liang T., Cascieri M., Cheung A., Brooks J. and Berman C.: Azasteroids: structure-activity relationships for inhibition of 5~-reductase and of androgen receptor binding. J. Med. Chem. 29 (1986) 2298-2315. Summerfield A. E., Diaz Cruz P. J., Dolenga M. P., Smith H. E., Strader C. and Toney J. H.: Tissue-specific pharmacology of testosterone and 5~-dihydrotestosterone analogues: characterization of a novel canine liver androgen binding protein. Molec. Pharmacol. 47 (1995) 1080-1088.
Pergamon
MK386: a P o t e n t , Selective Inhibitor o f the Human Type 1 5 -reductase K. Ellsworth, B. Azzolina, W. Baginsky, H. Bull, B. Chang, G. Cimis, S. Mitra, J. Toney, R. K. Bakshi, G. R. Rasmusson, R. L. Tolman and G. S. Harris* Departments of Enzymotogy and Medicinal Chemistry, Merck Research Laborawries, P.O. Box 2000, R80Y-140 Rahway N J 07065, U.S.A.
Steroid 5~-reductase is required for the c o n v e r s i o n o f testosterone to dihydrotestosterone. Loca!!zation o f type 1 5~-reductase in the s e b a c e o u s gland o f skin offers the possibility for selective i n h i b i t i o n o f this i s o z y m e as a t r e a t m e n t for ache. The goals o f these studies are to d e m o n s t r a t e the m e c h a n i s m o f i n h i b i t i o n o f MK386 a n d its selectivity for type 1 5~-reductase. The apparent p o t e n c y o f MK386 differed d e p e n d i n g o n the source o f the e n z y m e (i.e. r e c o m b i n a n t vs. native), yet selectivity for type 1 5~-reductase was u n c h a n g e d . O u r results indicate that the apparent p o t e n c y o f MK386 is m o d u l a t e d by the m e m b r a n e c o n c e n t r a t i o n o f the assay. T h e s e results suggest that MIO86 has a h i g h affinity for the lipid-rich m e m b r a n e e n v i r o n m e n t o f 5~-reductase. MK386 was also f o u n d to be a slow b i n d i n g inhibitor o f type 1 5~-reductase. However, the cause o f this t i m e - d e p e n d e n t i n h i b i t i o n is unrelated to partitioning o f the inhibitor into the m e m b r a n e b e c a u s e similar studies with type 2 5~-reductase indicate that MK386 is a reversible, c o m p e t i t i v e inhibitor. A n u m b e r o f c o u n t e r s c r e e n s were developed to d e m o n s t r a t e that MK386 is a p o o r inhibitor o f other steroid m e t a b o l i z i n g e n z y m e s . Copyright © 1996 Elsevier S c i e n c e Ltd.
J. Steroid Biochem. Mole,:. Biol., Vol. 58, No. 4, pp. 377-384, 1996
INTRODUCTION
inhibition of the type 1 enzyme for acne or hirsutism and inhibition of type 2 5~R for B P H or prostate cancer. T h e local conversion of testosterone (T) to the more It is now quite clear that selective inhibitors can be potent androgen 5~-dihydrotestosterone ( D H T ) by designed for type 1 and 2 5erRs. Potent, selective 5ct-reductase (5~tR) is implicated in various disease inhibitors of type 1 5~R have been reported, but no states, including benign prostatic hyperplasia (BPH), specificity data are available [6-9]. Finasteride and androgenetic alopecia, hirsutism and acne [1-3]. T h e epristeride (Fig. 1), type 2 5ctR selective inhibitors, have association of D H T with these disorders has generated undergone clinical testing for the treatment of great interest in 5ctR as a therapeutic target. However, B P H [10, 11]. Clinical studies with finasteride revealed recent identification of isozymes of 5ct R has complicated that serum levels of D H T are reduced by as m u c h as the search for clinically( useful inhibitors. T h e s e two 70%, whereas prostatic D H T decreased _> 90% at isozymes, 5~R type 1 and type 2, are differentially doses of 1 and 5 m g [12]. N o further decrease in expressed in tissues [4]. T h e type 1 enzyme is localized circulating D H T was observed at higher doses. T h e in scalp skin and liver whereas the type 2 enzyme is source of the residual circulating D H T in patients present in prostate, epididyrnis, seminal vesicles and treated with this agent is p r e s u m e d to be due to type 1 liver [4, 5]. T h e distinct tissue distribution of the 5~R, the finasteride-resistant form of the enzyme. In isozymes of 5ctR offers the possibility of selective support of this suggestion, recent studies indicate that maximal doses of M K 3 8 6 suppress serum D H T *Correspondence to: Dr G. S Harris. Tel.: + (908)594-7641; Fax: approximately 40% from baseline [9]. +(908) 594-5468. T h e high levels of type 1 5~R in scalp and other Abbreviations: T, testosterone, DHT, dihydrotestosterone, 5ctR, regions of skin as well as localization in sebaceous 5ct-reductase. Received 15 Nov. 1995; accepted 26 Jan. 1996. glands [13] are consistent with a proposal that the type 377
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K. Ellsworth et aL
1 enzyme is important in controlling the function of sebaceous glands. In this regard, a type 1 5~R inhibitor may offer potential for the treatment of androgenetic alopecia or acne. M K 3 8 6 (Fig. 1) was previously selected to evaluate the clinical utility of an inhibitor of type 1 5~R [8]. In this study we describe the kinetic properties of M K 3 8 6 and provide evidence for its selectivity for type 1 5~R.
MATERIALS
5~R assay
T h e assays of the native and recombinant h u m a n 5~Rs were performed as described previously [5, 8]. T h e baculovirus-expressed 5ctR was provided by K. Chan and D r S. Andersson [15]. Native sources of the human type 1 and 2 5~-reductases were human scalp and prostate, respectively. Kinetic analysis
Data from progress curves of product formation were fitted to the integrated first order equation (1) where y = product, vo is the initial velocity, v~ is the velocity at infinity time and k is the pseudo first order rate constant [16]. T h e second order rate constants ( k 3 / I ~ were estimated using equation (2) where k4 (the reverse H i
Finasteride
y = Vst = (Vo - v~) (I - e-k~)/k
(1)
k = k4 + k3(I/K(1 + S/Km + I/K~))
(2)
Vo= VmaxS/(Km(1 + I/KO + s).
(3)
AND METHODS
[7-3H]Testosterone, [14C]-acetate, [7-3H]-pregneno lone, [7-3H]cholesterol, [7-3H]-androstenedione and [ 1,2,4,5,6,7,16,17-3H]-dihydrotestosterone were purchased from New England Nuclear (Boston, MA). H u m a n tissues were obtained from the International Institute for the Advancement of Medicine (IIAM, Exton, PA). M K 3 8 6 (4,7-dimethyl-4-azacholestan-3one), L-733,690 (4,7-dimethyl-4-azacholestan-5-ene3-one), dihydrofinasteride, L-651,580, L-592,887 (6fl-hydroxy-20-spirox-4-ene-3-one) and L-603,075 (17fl-ureidoandros-4-en-3-one) were provided by Drs Raman Bakshi and Gary Rasmusson. All chemicals were obtained from Sigma Chemical Co. (St Louis, MO). Protein concentrations were estimated using the method developed by Lowry [14].
= H
rate constant) was taken to be zero because the inhibition curves plateau indicating the inhibition is pseudo-irreversible under these conditions. T h e K, for formation of the preliminary EI complex was determined using equation (3)
P a t h w a y assay for conversion o f acetate to cholesterol
T h e effects of M K 3 8 6 on the h u m a n hepatoma cell-line H e p G 2 were examined. H e p G 2 cells were plated in 6-well plates and grown overnight in D M E M supplemented with 10% serum. Cells were grown for an additional 24 h in the presence of 2 ml medium supplemented with 10% delipidated serum to maximize cholesterol biosynthesis. T h e cells were treated with M K 3 8 6 at various concentrations for 20 h. After 18 h, 5 mCi of 14C-acetate was added to each well. T h e cells were then extracted with 1 ml 1 N N a O H , saponified by heating at 95°C for 1 h, neutralized with HCI and extracted with 3 x 5 ml petroleum ether. T h e extracts were evaporated to dryness under a stream of nitrogen, resuspended in 1 0 0 / d ethyl acetate and applied to PE SIL G plates (Whatman, Clifton, N'J). Cholesterol was separated from other non-saponifiable lipid components by thin layer chromatography on silica gel plates using petroleum ether:diethyl ether:glacial acetic acid (75:25:1). T h e incorporation of 14C into cholesterol and squalene was quantified using the phosphorimager and Imagequant application software. A s s a y for the conversion of cholesterol to pregnenolone
H u m a n adrenal tissue was pulverized with a freezer mill and resuspended in three volumes of 25 m M Tris, p H 7.4, 1 m M E D T A , 250 m M sucrose and 20% glycerol. T h e suspension was homogenized with a Dounce homogenizer and the crude homogenate used as the source of enzyme in this assay. T h e reaction mixture contained 35 m M sodium phosphate p H 7.4, 20 m M potassium chloride, 5 m M magnesium chlor-
H
HOOC
! CH3 Epristeride
Fig. 1. Inhlbitors o f 5~-reductase.
MK386
MK386: A Potent, Selective Inhibitor of the Human Type 1 5~-Reductase ide, 0.2 m M
E D T A , 2 0 0 m M sucrose, 6 p M [710 m M isocitrate, and 1 m M N A D P H in a final volume of 50 pl. M K 3 8 6 was added to the assay mixture to give a tinal concentration of 1000 n M in 0.5% ethanol. L-592,887 (6fl-hydroxy-20-spirox-4ene-3-one), an inhibitor of the side chain cleavage enzyme, was included a:~ a positive control in the assay at a concentration of 50 pM. T h e reaction was initiated by the addition of adrenal homogenate and incubated for 2 h at 37°C. T h e mixture was quenched with cyclohexane:ethyl acetal:e (36:64 v/v) and the aqueous and organic layers were separated by centrifugation at 14,000 rpm in an E p p e n d o r f microfuge. T h e organic layer was subjected to normal phase H P L C (25 cm Whatman partisil 5 silica column equilibrated in 1.2 ml/min cyclohexane:ethyl acetate (36:64 v/v), retention times cholesterol, 5.4 min; pregnenolone, 8 min).
3H]cholesterol,
Pathway assay for the conversion of pregnenolone to 5-androstenediol H u m a n testicular tissue was pulverized in liquid nitrogen using a freezer :mill and homogenized in 1 m M M O P S p H 7.2, 100 m M potassium chloride, 1 m M E D T A , 2 m M D T T containing 1 M sucrose using a Potter-Elvehjem homogenizer. This crude homogenate was used as the source of enzymes in this assay. T h e reaction mixture contained 40 m M potassium phosphate, p H 7.5, 0.09 # M [7-3H]-pregnenolone, 1 m M D T T , 0.5 p M N A D P + and 3.6 p M NAD÷in a final volume of 100 pl. M K 3 8 6 was added to the assay mixture to give a final concentration of 1 n M - 1 0 # M inhibitor in 0.5% ethanol. L-603,075 (17fl-ureidoandros-4-en-3-one), an inhibitor of the C17,20lyase, was added as a positive control in this assay. T h e reaction was initiated by the addition of testicular homogenate corresponding to 3 mg ]protein and incubated at 37°C. After 10 min the reaction was quenched by extraction with 290 pl of cyclohexane:ethyl acetate (70:30 v/v) containing 10 #g each unlabelled dehydroepiandrosterone (DHEA), androstenedione and testosterone. T h e organic layer was subjected to normal phase H P L C (25 cm W h a t m a n partisil 5 silica column equilibrated in 1.2 ml/min cyclohexane:ethyl acetate (80:20 v/v), retention times pregnenolone, 12 min; 17ct-hydroxypregnenolone, 7.5 min; dehydroepiandrosterone, 15 min; 5-androstenediol, 20 min). T h e conversion of pregnenolone to 17~-hydtoxypregnenolone by 17~-hydroxylase, subsequent conversion to D H E A by 17=~-hydroxypregnene (;1720 lyase and further conversion to 5-androstenediol by 17fl-hydroxysteroid dehydrogenase was monitored using the radioactivity flow detector. Following a 10 rain incubation under the assay conditions described, the product distribution was 50% pregnenolone, 5% 17~-hydroxypregnenolone, 10% dehydroepiandrosterone and 23% 5-androstenediol.
379
Aromatase assay A sample of h u m a n placenta was homogenized in two volumes 1 m M M O P S , p H 7.2, 100 m M potassium chloride, 1 m M E D T A , 2 m M D T T and 1 M sucrose using a Brinkmann Instruments Polytron followed by standard D o u n c e homogenization (15 x). T h e sample was centrifuged at 1 0 , 0 0 0 x g for 10 min, the supernatant was collected and centrifuged at 100,000 x g for 1 h at 4°C. T h e microsomal pellet was washed once and resuspended in buffer and stored frozen at -80°C. T h e reaction mixture for the aromatase assay contained 50 m M potassium phosphate, p H 7.5, 500 # M N A D P H , 0.5 m M E D T A , 0.5 m M D T T , 40 n M [7-3H]-androstenedione, 5 m M magnesium chloride and 3.1 pg h u m a n placental microsomes in a final volume of 100 pl. T h e assay was initiated by the addition of enzyme and incubated at 37°C. M K 3 8 6 was added to the assay mixture to give a final concentration of 1 n M - 1 0 # M in 0.5% ethanol. A T D (1,4,6-androstatriene-3,17-dione), a known inhibitor of aromatase was included as a positive control in the assay. After 20 min the reaction was quenched by extraction with 250#1 of a mixture of cyclohexane:ethyl acetate (64:36v/v) containing 1 0 p g each T , D H T and androstenedione. T h e organic layer was subjected to normal phase H P L C (25 cm Advantage Plus 5 micron silica column equilibrated in 1.2 ml/min cyclohexane: ethyl acetate (70:30v/v); retention times estrone, 5.0 min; estradiol, 7.5 min; androstenedione, 11 min. T h e conversion of androstenedione to estrone was monitored using the radioactivity flow detector by mixing the H P L C effluent with two volumes of Flo Scint II (Radiomatic, T a m p a , FL).
17fl-hydroxysteroid dehydrogenase Samples of liver were pulverized using a freezer mill and homogenized in 40 m M potassium phosphate, p H 6.5, 5 m M magnesium sulphate, 25 m M potassium chloride, 1 m M phenylmethylsulfonyl fluoride, 1 m M D T T containing 0.25 M sucrose using a Potter-Elvehjem homogenizer. T h e sample was centrifuged at 11,500 x g for 15 min. T h e supernatant from this step was collected and subjected to a final centrifugation at 105,000 x g for 1 h at 4°C. T h e microsomal pellet was washed two times and resuspended in two volumes of buffer. Glycerol was added to the resuspended pellet to a final concentration of 20%. T h e reaction mixture contained 40 m M potassium phosphate, p H 7.5, 2 m M N A D ÷, 1 m M E D T A , 5 m M D T T and 2 # M [7-3H]-T in a final volume of 100 pl. M K 3 8 6 was added to the assay mixture to give a final concentration of 1 n M - 1 0 # M in 0.5% ethanol. T h e assay was initiated by the addition of 0.4 #g human liver microsomes and incubated at 37°C. After 20 rain the reaction was quenched by extraction with 250 pl of a mixture of cyclohexane:ethyl acetate (64:36v/v) containing 10 #g each T, D H T and androstenedione.
380
K. Ellsworth et al.
T h e organic layer was subjected to normal phase H P L C (25 cm Advantage Plus 5 micron silica column equilibrated in 1.2 ml/min cyclohexane: ethyl acetate (64:36 v/v); retention times androstenedione, 8.5 min; testosterone, 11.5 min). T h e conversion of T to androstenedione was monitored using the radioactivity flow detector by mixing the H P L C effluent with two volumes of Flo Scint II (Radiomatic).
Table 1. Inhibition of 5ct-reductase
ICs0 (nM) Inhibitor MK386 MK386 Dihydrofinasteride Dihydrofinasteride L-651,580 L-651,580
Enzyme source
Type 1
Type 2
Native Recombinant Native Recombinant Native Recombinant
20 0.9 700 540 40 43
3100 150 15 6 960 720
H u m a n androgen receptor assay
T h e stable transfectant cell line expressing the h u m a n androgen receptor (hAR) was provided by Prof. Michael McPhaul at the Southwestern Medical School in Dallas, T X [17]. T h e cell line was maintained in H a m ' s F12 m e d i u m (Gibco, G r a n d Island, NY) supplemented with 10% fetal calf serum (Gibco), P S N antibiotic mixture (Gibco) and 0 . 4 m g / m l G418 (Gibco). T h e cells are fed three times weekly, and are maintained at 37°C with 5% CO2. Cells are grown to near confluence, and are washed twice with PBS (phosphate buffered saline). T h e cells are harvested and the pellet is washed twice with T E G M ( 1 0 m M Tris-HC1; 1 m M E D T A ; 10% glycerol; 1 m M beta-mercaptoethanol, 10 m M sodium molybdate; p H 7.2) buffer. T h e cell suspension was frozen in liquid nitrogen and stored at -80°C. Prior to the assay, the suspension was thawed at 4°C and centrifuged for 1 h at 45,000 r p m (Ti70) at 4°C to remove cellular debris. [1,2,4,5,6,7,16,17-3H]DHT was added to the extract (supematant) to give a final concentration of 1 nM. Typically the assays were done in triplicate by dispersing 0.1 ml of the s u p e m a t a n t into borosilicate glass tubes containing inhibitor. T h e final concentration (v/v) of ethanol does not exceed 1% in the assay. T h e mixture was incubated overnight at 4°C. Free [ 3 H ] D H T was separated from receptor-bound h o r m o n e by the addition of 0.1 ml of dextran coated charcoal (0.5% dextran T - 7 0 , 0.1% gelatin; 5% charcoal in 10 m M Tris, p H 7.0, 0.1 m M E D T A ) . T h e suspension was centrifuged for 15 min at 5000 rpm. T h e 3H in the supernatant was determined by scintillation counting and represented the receptorbound DHT.
RESULTS
Inhibition o f the h u m a n type 1 and 2 5c~R
Initial studies to identify a selective inhibitor of 5~R were carried out using h u m a n scalp and prostate as sources of the h u m a n type 1 and 2 5c~Rs, respectively. T h e results of these analyses led to the discovery of M K 3 8 6 , a 4-N-methyl azasteroid [8]. As indicated in Table 1, M K 3 8 6 is a potent inhibitor of type 1 5c¢R in h u m a n scalp skin with an IC50 of ,,~ 20 nM. This c o m p o u n d is a poor inhibitor of type 2 5~R in h u m a n prostate with an IC~0 of ~ 3100 nM. These results indicate that the selectivity is > 150-fold for type 1
5~R. Similar selectivity is found using recombinant type 1 and 2 expressed in baculovirus, although the ICs0 values for M K 3 8 6 are ~ 20-fold lower than those obtained using the native enzymes (Table 1). Interestingly, no differences are noted in ICs0 values for dihydrofinasteride or L-651,580 which are two standards used in the assays to compare native and recombinant 5ctR. T h e difference in IC~0 values for M K 3 8 6 with native and recombinant 5~Rs was unexpected, and appeared to be restricted to the aza-cholestan-3-one class of inhibitors. Additional experiments were designed to explore the cause of the apparent potency differences with MK386. One obvious feature of this c o m p o u n d to consider is the hydrophobicity. It is reasonable to assume that M K 3 8 6 preferentially binds to protein or partitions into the m e m b r a n e rather than behave as a freely soluble inhibitor. Any effect of protein binding on the apparent potency can be ruled out since ICs0 values for the native enzyme were unchanged in the presence of 20 mg/ml BSA or 50% fetal calf serum (data not shown). A more likely possibility is that the hydrophobicity of the c o m p o u n d leads to significant partitioning into the m e m b r a n e , ultimately leading to a decrease in the solution concentration and an increase in the m e m b r a n e concentration. In this case, the higher specific activity of the recombinant enzyme results in significantly lower amounts of m e m b r a n e in the assay mixture c o m p a r e d to the native enzyme. I f the difference in m e m b r a n e concentration in the assay mixture gives rise to the discrepancy in the IC~0 values for M K 3 8 6 with native and recombinant 5~R, then addition of control (non-5~R containing) m e m b r a n e s should alter the apparent potency of M K 3 8 6 while inhibition of the enzyme by the standard c o m p o u n d s should not be affected. In order to test the m e m b r a n e partitioning hypothesis, the ICs0 for M K 3 8 6 for the recombinant type 2 5~R was determined in the presence of variable amounts of control m e m b r a n e s prepared from uninfected baculovirus. As listed in Table 2, the ICs0 for M K 3 8 6 increased with increasing amounts of m e m b r a n e , approaching those obtained with native type 2 5~R. T h e same dependence of ICs0 value on m e m b r a n e concentration was observed with L-733,690, a related 4-aza-cholestan-3-one. As predicted, IC~0 values in control experiments with
MK386: A Potent, Selective Inhibitor of the Human Type 1 5~t-Reductase dihydrofinasteride and L-651,580 are not altered as a function of the membrane concentration. F r o m these experiments, we conc/'ade that the potency differences observed with native and recombinant 5~tR are not due to differences in the enzyme, but simply reflect changes in the membrane concentration in the assay mixture. At present it is unclear whether the enzyme is exposed to the aza-cholestan-3-ones from the membrane or the solution.
11.0 l
7.3
n
Mechanism of inhibition
3.7
As illustrated in Fig. 2A, progress curves of D H T formation by native type 1 5~R are non-linear in the presence of M K 3 8 6 and indicate that this inhibitor is more potent than predicted from a fixed time point ICs0 determination. Similar results were obtained using either native or recombinant enzyme; therefore, the time-dependent inhibition does not reflect membrane influences on potency but direct interaction with the enzyme. T h e data in Fig. 2A, obtained using native type 1 5~R, indicate this inhibitor is slow binding and forms an initial complex with a Ki= 14 n M which then rearranges to a high affinity complex with a pseudo first order rate constant of 3 x 10 -3 s-L T h e second order rate constant is kJKi = 2.5 x 105 M - ~s - 1. These data are consistent with slow-binding inhibition as described by Morrison and Walsh [16] where inhibitor binds to enzyme to produce a loosely associated Michaelis complex EI which isomerizes to give EI*, a tightly b o u n d complex (Fig. 1) which for M K 3 8 6 gives a dissociation constant Scheme <100 pM. Although a similar kinetic scheme had been described for the inhibition of 5~R by finasteride [18, 19], the mechanism of inhibition for M K 3 8 6 is different because it is unable to undergo the reduction step as found with finasteride [20]. Ki
Scheme 1
381
It3
F, + I ~.~ E:I ~ E:I* k4
In contrast to that found for native type 1 5~R, the time course for inhibition for native type 2 5~R by M K 3 8 6 indicates that this c o m p o u n d is a classic reversible inhibitor of dais enzyme (Fig. 2b). In this Table 2. Influence of membrane concentration on inhibition of Oepe 2 5M~! by MK386 IC~0 (nM) Enzyme source control microsomes
r5~R 0 pg
r5~R 0.9 p g
r5~R 45 p g
Native 5c~R
Compound MK386 L-733,690 Dihydrofinasteride L-651,580
63 100 6 630
75 240 7 780
700 5000 4 700
3100 > 1000 15 960
Assays were carried out as described in M e t h o d s with type 2 5~R produced recombinantly (r5~R) using baculovirus or from h u m a n prostate (native) as described previously [5]. Control microsomes were obtained from n o n - 5 ~ R producing baculovirus a n d were included in the 100 #1 assay as indicated.
•
:
8
24 nM I
00
J
42
84
126
min 19.0 Type 2
/ aM
12.7 -
"0
.o
6.4
00 °
I
1
42
84
126
min
Fig. 2. I n h i b i t i o n o f 5 ~ - r e d u c t a s e b y M K 3 8 6 . A , R e a c t i o n p r o g r e s s c u r v e s for n a t i v e t y p e 1 5 ~ - r e d u c t a s e i n a s s a y m i x t u r e c o n t a i n i n g 0.1 M p o t a s s i u m p h o s p h a t e , p H 7.2, 5 / i M [7-3H]T , 5 0 0 / ~ M N A D P H w i t h M K 3 8 6 as i n d i c a t e d . B , R e a c t i o n p r o g r e s s for n a t i v e t y p e 2 5 ~ - r e d u c t a s e in an a s s a y m i x t u r e c o n t a i n i n g 40 m M s o d i u m citrates p H 5.5, 0 . 3 / * M [ 7 - ' H ] - T , 5 0 0 / ~ M N A D P H a n d M K 3 8 6 as i n d i c a t e d .
case, additional mechanistic studies are more straightforward and are not complicated by the time-dependent nature of the inhibition as found with type 1 5ctR. In order to minimize the effective dilution of inhibitor and consequently obtain the best estimate of the intrinsic potency of the c o m p o u n d , recombinant 5~t-reductase type 2 was used as the source of enzyme in these studies. As indicated in Fig. 3, M K 3 8 6 exhibits competitive inhibition of type 2 5~tR vs. T with an apparent / ~ = 9 0 nM. Therefore, despite the influence of the membrane concentration on the apparent potency, the inhibition of 5~R by M K 3 8 6 is not a consequence of altering the membrane environment of the enzyme, but is due to binding at the enzyme active site.
382
K. Ellsworth et aL 5.7
4.6
2.3 3.4
g
1.1
I
3.0
I
I
6.0 9.0 I/[Testosterone] (gM -1)
I
12.0
Fig. 3. C o m p e t i t i v e i n h i b i t i o n o f t h e r e c o m b i n a n t t y p e 2 5 ~ - r e d u c t a s e b y M K 3 8 6 . T h e e n z y m e w a s i n c u b a t e d in a m i x t u r e c o n t a i n i n g 40 m M s o d i u m c i t r a t e , p H 5.5, 5 0 0 / i M N A D P H , 0.3/~M [7-3H]-T a n d M I O 8 6 a s i n d i c a t e d .
Selectivity of M K 3 8 6
Lack of effect on the conversion of cholesterol to pregnenolone
Previous work in the azasteroid series demonstrated that additional activities were associated with some of the 4-N-methyl analogs. Most notably, 4 M A (17flN,N-diethylcarbamoyl-4-methyl-4-aza-5~-androstan3-one) was found to interact with the rat androgen receptor [21], as well as to inhibit the bovine adrenal 3fl-hydroxy-5-ene-steroid dehydrogenase/3-keto-5-enesteroid isomerase [22]. Given the structural similarity of the A ring of M K 3 8 6 to 4MA, it was prudent to demonstrate the specificity of M K 3 8 6 for type 1 5~R. A n u m b e r of counterscreens were developed to test the selectivity of this c o m p o u n d with the h u m a n enzymes. Several pathway assays were developed to monitor potential effects of M K 3 8 6 on enzymes involved in the overall conversion of acetate to cholesterol, as well as further conversion to testosterone or estrogens. These pathway assays facilitate the survey of multiple enzymes in a single mixture. Inhibition of any of these steps is unacceptable in a candidate slated for clinical development because of potential effects on the formation of testosterone, glucocorticoids, mineralocorticoids or estrogens.
T h e conversion of cholesterol to pregnenolone requires the action of the C20-hydroxylase, C22-hydroxylase and the C20,22 side chain cleavage enzyme. Although M K 3 8 6 is a 4-azasteroid, the cholesterol side chain at C,7 could permit this c o m p o u n d to act as an alternate substrate or inhibitor of these enzymes and potentially block the initial steps in the biosynthesis of all steroid hormones. In order to determine whether M K 3 8 6 is an inhibitor of these enzymes, an assay was developed to monitor the conversion of radiolabelled cholesterol to pregnenolone using h u m a n adrenal homogenate, a major site of steroid h o r m o n e biosynthesis. T h e results of these studies indicate that at 10,000 n M M K 3 8 6 there is no effect on the production of pregnenolone (Table 3). Lack of effect on the conversion of pregnenolone 5-androstenediol
to
As indicated above, M K 3 8 6 is a 4-azasteroid which could potentially inhibit other steroid metabolizing enzymes besides 5~R. In order to determine whether this c o m p o u n d inhibits 17~-hydroxylase, 17a-hydroxyTable 3. Selectivity of MK386 for type 1 5c~-reductase
Lack of effect on the conversion of acetate to cholesterol
Enzyme/pathway
ICso (nM)
An assay in H E P G 2 cells was used to monitor the overall conversion of acetate to squalene or cholesterol. Cells were grown in the presence and absence of M K 3 8 6 , in the presence of delipidated serum to upregulate cholesterol biosynthesis and then supplemented with [14C]acetate. T h e incorporation of 14C into squalene and cholesterol was monitored. M K 3 8 6 is a p o o r inhibitor of squalene and cholesterol biosynthesis with ICs0 values of 5 0 0 0 n M and 10,000 nM, respectively (Table 3).
5g-reductase type 1' 5g-reductase type 2 a Acetate ~ squalene Acetate ~ cholesterol Cholesterol ~ pregnenolone Pregnenolone --, 5-androstenediol 17fl-hydroxysteroid dehydrogenase type 2 Aromatase 3fl-hydroxysteroid dehydrogenase/isomerase Androgen receptor
< 20 3100 5000 10,000 > 1000 > 10,000 > 10,000 > 10,000 8000 > 100,000
aValues obtained using native sources of 5~-reductase.
MK386: A Potent, Selective Inhibitor of the Human Type 1 5ct-Reductase p r e g n e n e C17,20-1yase o r t y p e 3 1 7 f l - h y d r o x y s t e r o i d dehydrogenase, a modification of the system developed b y Y a n a i h a r a a n d T r o e n [23] was u s e d to m o n i t o r t h e c o n v e r s i o n o f r a d i o l a b e l l e d p r e g n e n o l o n e to 5 - a n d r o s t e n e d i o l . H u m a n t e s t i c u l a r tissue, a m a j o r site o f a n d r o g e n b i o s y n t h e s i s , was u s e d as a s o u r c e o f t h e s e e n z y m e s for these assays. M K 3 8 6 h a d n o effect o n the c o n v e r s i o n o f p r e g n e n o l o n e to 5 - a n d r o s t e n e d i o l w h e n t e s t e d u p to 10,000 n M ( T a b l e 3). T h e r e f o r e , M K 3 8 6 does not inhibit the human testicular 17~-hydroxylase, 17~-hydroxypregnene (217_20 lyase or 1 7 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e . I n c o n t r a s t , L - 6 0 3 , 0 7 5 (17flureidoandros-4-en-3-one) a compound previously r e p o r t e d as an i n h i b i t o r o f t h e C17,20-1yase [24] a p p e a r e d to b l o c k t h e 1 7 ~ - h y d r o x y l a s e a n d r e s u l t e d in 8 4 % i n h i b i t i o n o f the p a t h w a y at 10,000 n M . L a c k of inhibition o f 3fl-hydroxysteroid dehydrogenase/isomerase P r e v i o u s studies b y B r a n d t a n d L e v y [22] i n d i c a t e t h a t several 4 - N - m e t h y l a z a s t e r o i d s are p o t e n t i n h i b i tors o f t h e b o v i n e 3 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e / i s o m e r a s e w i t h low n M K, values. T o date, little w o r k has b e e n d o n e to s t u d y t h e i n h i b i t i o n o r s t r u c t u r e - a c tivity r e l a t i o n s h i p o f t:he h u m a n 3 f l - h y d r o x y s t e r o i d dehydrogenase/isomerase by 4-azasteroids. These studies w e r e i n i t i a t e d to d e t e l a n i n e the affinity o f M K 3 8 6 for human type 1 3fl-hydroxysteroid dehydrogenase/isom e r a s e f r o m h u m a n p l a c e n t a , a n essential e n z y m e in t h e f o r m a t i o n o f m a n y o f the s t e r o i d h o r m o n e s . M K 3 8 6 is a p o o r i n h i b i t o r o f t y p e 1 3 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e / i s o m e r a s e a n d is n o t e x p e c t e d to effect the production of glucocorticoids, mineralocorticoids, p r o g e s t i n s o r a n d r o g e n s ( T a b l e 3). L a c k o f inhibition o f 17fl-hydroxysteroid dehydrogenase T h e i n t e r c o n v e r s i o n o f a n d r o s t e n e d i o n e to t e s t o s t e r o n e a n d e s t r o n e to e s t r a d i o l is c a t a l y s e d b y 17fl-hyd r o x y s t e r o i d d e h y d r o g e n a s e . M u l t i p l e i s o z y m e s o f this e n z y m e exist w h i c h m a y differ in sensitivity to M K 3 8 6 . Results obtained using human testicular homogenates to m o n i t o r the c o n v e r s i o n o f p r e g n e n o l o n e to 5 - a n d r o s t e n e d i o l i n d i c a t e t h a t t h e r e is n o i n h i b i t i o n o f t h e t e s t i c u l a r (type 3) 1 7 f l - h y d r o x y s t e r o i d d e h y d r o g e n ase. A s e c o n d assay was d e v e l o p e d to d e t e r m i n e the effects o f M K 3 8 6 o n t y p e 2 1 7 f l - h y d r o x y s t e r o i d d e h y d r o g e n a s e , t h e form f o u n d in h u m a n liver [25]. N o i n h i b i t i o n o f this isofonaa was o b s e r v e d w i t h M K 3 8 6 ( T a b l e 3). L a c k of inhibition o f aromatase T h e p o t e n t i a l use o f a t y p e 1 5c~R i n h i b i t o r for the t r e a t m e n t o f a c n e or h i r s u t i s m in w o m e n r e q u i r e s t h a t t h e r e b e n o effects o n the b i o s y n t h e s i s o f estrogen. N o i n h i b i t i o n o f h u m a n p l a c e n t a l a r o m a t a s e was o b s e r v e d in t h e p r e s e n c e o f M K 3 8 6 (ICs0 > 10,000 n M , T a b l e 3). T h e r e f o r e , this c o m p o u n d s h o u l d n o t interfere w i t h t h e p r o d u c t i o n o f e s t r o g e n !in w o m e n .
383
N o detectable binding to the h u m a n androgen receptor M o s t o f t h e studies to define the s t r u c t u r e activity r e l a t i o n s h i p for b i n d i n g o f a z a s t e r o i d s to the a n d r o g e n r e c e p t o r have f o c u s e d o n the r e c e p t o r f r o m rat r a t h e r t h a n h u m a n [26]. T h e r e f o r e , d e s p i t e t h e w e a l t h o f i n f o r m a t i o n d e s c r i b i n g t h e b i n d i n g c h a r a c t e r i s t i c s o f the r a t a n d r o g e n r e c e p t o r , little i n f o r m a t i o n was available to p r e d i c t w h e t h e r M K 3 8 6 w o u l d b i n d to t h e h u m a n a n d r o g e n r e c e p t o r . I n o r d e r to o v e r c o m e this l i m i t a t i o n , a s c r e e n i n g assay was d e v i s e d u s i n g t h e c l o n e d h u m a n a n d r o g e n r e c e p t o r [17, 27]. R e c e p t o r b i n d i n g assays o f M K 3 8 6 r e v e a l e d t h e r e is n o d e t e c t a b l e b i n d i n g to the c l o n e d h u m a n r e c e p t o r ( T a b l e 3). T h i s r e s u l t i n d i c a t e s t h e r e is > 5 0 0 0 - f o l d selectivity for t y p e 1 5 ~ R c o m p a r e d to the h u m a n a n d r o g e n r e c e p t o r .
CONCLUSION
T h e results d e s c r i b e d in this p a p e r i n d i c a t e t h a t M K 3 8 6 is a p o t e n t i n h i b i t o r for h u m a n t y p e 1 5ciR. T h e l a c k o f effect o f M K 3 8 6 in any o f the c o u n t e r s c r e e n s suggests that, w h e n u s e d clinically, M K 3 8 6 is selective for t h e t a r g e t e n z y m e , t y p e 1 5~R. T h e availability o f this i n h i b i t o r d e f i n e d t h e c o n t r i b u t i o n o f t y p e 1 5 ~ R to c i r c u l a t i n g D H T levels in h u m a n s [9]. G i v e n t h e l o c a l i z a t i o n o f t h e t y p e 1 i s o z y m e in t h e s e b a c e o u s g l a n d a n d t h e l i p i d - r i c h s e c r e t i o n o f the g l a n d , t h e h y d r o p h o b i c p r o p e r t i e s o f the i n h i b i t o r m a d e it an attractive c a n d i d a t e for the t r e a t m e n t o f acne.
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