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Inhibition of rat α-reductases by finasteride: Evidence for isozyme differences in the mechanism of inhibition
J. Steroid Biochem. Molec. Biol. Vol. 61, No. 1/2, pp. 55-64, 1997 ~.~ 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S 0 9 6 0 - 0 7 6 0 ( 9 7 ) 0 0 0 0 2 - 2 0960-0760/97 $17.0o + o.0o
Pergamon
I n h i b i t i o n of Rat -Reductases by Finasteride: E v i d e n c e for Isozyrne D i f f e r e n c e s in the M e c h a n i s m o f Inhibition B. A z z o l i n a , ' K. E I I s w o r t h , 1 S . A n d e r s s o n , 2 W. G e i s s l e r , ' H . G. Bull' a n d G. S. H a r r i s 1. IDepartment of Enzymoi!ogy, Merck Research Laboratories, PO Box 2000, R80Y-140, Rahway, N J 07065, U.S.A. and 2Department of Obsi~,etrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, T X 75235, U.S.A.
T h e m e c h a n i s m o f ird~ibition o f t h e r a t t y p e s 1 a n d 2 5 ~ - r e d u c t a s e b y f i n a s t e r i d e w a s i n v e s t i g a t e d u s i n g r e c o m b i n a n t l y e x p r e s s e d e n z y m e s . T h e s e s t u d i e s r e v e a l e d t h a t f i n a s t e r i d e is a p o t e n t , r e v e r s ible i n h i b i t o r o f t h e rat t y p e 1 5 ~ - r e d u c t a s e w i t h Ki = 10.2 _+ 1.3 n M . F i n a s t e r i d e is a p o t e n t i n h i b i t o r o f t h e r a t t y p e 2; h o w e v e r , in t h i s c a s e t h e c o m p o u n d b i n d s to t h e t y p e 2 i s o z y m e - N A D P H complex to f o r m a t e r n a r y c o m p l e x w i t h Ki = 1.19 + 0.10 n M , w h i c h t h e n r e a r r a n g e s to a h i g h a f f i n i t y c o m p l e x (E:I*) w i t h a p s e u d o first o r d e r rate c o n s t a n t o f 1.62 + 0.22 × 10-3Is. T h e s e c o n d o r d e r r a t e c o n s t a n t is kflKi = 1.37 + 0.31 × 10 6 MJs. H e a t d e n a t u r a t i o n o f t h e ( t y p e 2 e n z y m e : i n h i b i t o r ) c o m p l e x r e l e a s e s d i h y d r o f i n a s t e r i d e a n d p r e s u m a b l y t h e N A D P + - a d d u c t p r e v i o u s l y i d e n t i f i e d w i t h t h e h u m a n 5~r e d u c t a s e s . T h e e f f e c t s o f f i n a s t e r i d e w e r e a l s o s t u d i e d in i n t a c t C O S ceils t r a n s i e n t l y e x p r e s s i n g t h e rat t y p e s 1 a n d 2 5 ~ - r e d u c t a s e . R e s u l t s w i t h w h o l e cell a s s a y s c o n f i r m d i f f e r e n c e s in m e c h a n i s m o f i n h i b i t i o n o f r a t t y p e s 1 a n d 2 5 ~ - r e d u c t a s e b y f i n a s t e r i d e . © 1997 E l s e v i e r S c i e n c e Ltd
J. Steroid Biochem. Molec. Biol., Vol. 61, No. 1/2, pp. 55-64, 1997
INTRODUCTION
useful inhibitors for the treatment of benign prostatic hyperplasia and male pattern baldness began shortly after the discovery of 5~R. Beginning in the 1980s, many inhibitors of 5~R were described as potential clinical candidates. These compounds include finasteride (17fl-(N-tert-butylcarbamoyl)-4-aza-5~-androst-l-en-3-one, M K 9 0 6 ) [6,7], epristeride [8, 9], FK143 [10], turosteride [11], and the 6-azasteroids [12, 13]. Most inhibitors were discovered by evaluating the in vitro activity of compounds on the 5~R activity in extracts of human prostate. Subsequent in vivo studies were often conducted in a castrate rat model to evaluate the ability of compounds to block the T-stimulated growth of the prostate. T h e predictive clinical value of this rat model depended on the presumed similarity of the h u m a n and the rat forms of 5~R. Finasteride has been used as a prototype 5~R inhibitor by many investigators to define the effects of the inhibition of this enzyme in various rat models. Brooks et al. [14] demonstrated that this inhibitor prevented T - but not D H T - s t i m u l a t e d growth of the prostate in castrated male rats. These results confirm
Steroid 5~-reductase (5~R) is the N A D P H - d e p e n d e n t enzyme required for the conversion of testosterone (T) to dihydrotestosterone ( D H T ) . This enzyme is found in androgen target tissues such as the prostate, seminal vesicle and skin, which preferentially respond to D H T rather than to T [1]. T h e production of D H T is required for the normal development of the external genitalia of tile male fetus, thus defects in this enzyme give rise to a form o f male pseudohermaphroditism where affected males have ambiguous genitalia at birth [2, 3]. Post puberty, the consequences of life-long decreases in circulating D H T are an underdeveloped prostate and little or no male pattern baldness or acne [4, 5]. These findings led to the suggestion that D H T is the androgen which supports growth of the prostate and leads to scalp hair recession [4]. As a result, the search for clinically *Correspondence to Georgianna Harris. Received 13 Aug. 1996; accepted 26 Nov. 1996. Abbreviations: 5~R, 5~-reductase; T, testosterone; D H T , drotestosterone; PBS, phosphate buffered saline
dihy55
56
B. Azzolina et al.
that finasteride does not interact with the androgen receptor but blocks the formation of D H T to inhibit the growth of the prostate. Finasteride has also been used to define the effects and critical period in gestation during which 5ctR inhibition disrupts the normal development of male rats due to the need for D H T in the differentiation of the prostate and external genitalia [15-17]. Extrapolation of these results to h u m a n male development suggests that this critical period for the action of D H T is weeks 8 - 1 2 of gestation [15, 16]. T h e continued use of rat models to support the development of 5aR inhibitors is now complicated by the identification of two genes encoding two types of 5~R in the rat, m o n k e y and h u m a n [18-21]. Despite sequence homology between the rat and h u m a n isozymes, the tissue distribution and inhibitor sensitivity varies considerably between these two species [6, 20, 22, 23]. As a result of these issues, some investigators have pursued primate models to 5~R inhibitors [21, 24-26]. Given the ongoing use of finasteride as a 5~R inhibitor in various rat models, a thorough understanding of the m e c h a n i s m of inhibition of rat types 1 and 2 5~R is required to interpret the results of these in vivo studies fully. Recently, slow-binding inhibition of h u m a n types 1 and 2 5~R by finasteride was described [27, 28]. It is now clear that the mechanism of inhibition of the 5~Rs involves reduction of the Aring of the azasteroid and subsequent adduct formation with N A D P + (Fig. 1) [29]. T o date, little attention has been devoted to studying the inhibition of the rat 5~Rs. In this manuscript we describe the mechanism of inhibition of the rat isozymes of 5~R by finasteride. Furthermore, we describe the utility of intact C O S cells expressing 5~R as an alternative to crude enzyme preparations for the mechanistic study of inhibitors of this enzyme. MATERIALS AND METHODS
[7-3H]-Testosterone was purchased from N e w England Nuclear (Boston, MA, U.S.A.). Finasteride (was provided by D r Gary Rasmusson. [1,2-3H] Finasteride was prepared by D r Avery Rosegay, M e r c k Research Laboratories, by reduction of A 1 of unlabelled c o m p o u n d with tritium gas followed by reoxidation to reintroduce the double b o n d into the A ring [30]. All chemicals were obtained from Sigma Chemical Co (St Louis, M O , U.S.A.). Protein concentrations were estimated using the m e t h o d developed by Lowry et al. [31]. Expression of 5~R
R e c o m b i n a n t plasmid expressing the rat type 1 5~R was a generous gift from D r David Russell. Rat type 2 c D N A was cloned by reverse-transcriptase-polymerase chain reaction ( R T - P C R ) . Total R N A (Promega,
Madison, WI, U.S.A.; total isolation kit) was prepared from epididymis dissected from 2-month-old (sexually mature) S p r a g u e - D a w l e y rats. After reverse transcription, type 2 5aR was P C R amplified using specific primers corresponding to bases 3 8 - 5 7 in the 5' end and 8 5 4 - 8 7 2 in the 3' end of the c D N A [20]. T h e primers were designed so that the amplification product would contain the entire coding region, flanked by B a m H 1 and H i n d I I I sites. T h e P C R product of 856 base pairs was cloned into the m a m m a lian expression vector p C M V 6 [32] using the B a m H 1 and H i n d I I I sites. Double-stranded D N A sequencing confirmed the identity of the rat type 2 5~R cDNA. C O S cells were grown in Dulbecco's m i n i m u m essential m e d i u m ( D M E M ) (Gibco BRL, G r a n d Island, NY, U.S.A.) supplemented with 10% fetal bovine serum (Hyclone, Logan, U T , U.S.A.) and transfected on day 0 by electroporation using 20 #g plasmid D N A and 10 7 cells per cuvette. Whole cell assays
C O S cells were grown and transfected as described. Control cells were m o c k transfected and handled under the same conditions. On day 2-3, the m e d i u m was removed, replaced with phosphate buffered saline (PBS) containing 1 0 0 n M [3H]-T for type 1 and 50 n M for type 2 5~R. Samples of buffer were withdrawn at various times and the steroids extracted with a mixture of cyclohexane:ethylacetate (55:45v/v). Separation of T and 5~-reduced products was accomplished by normal phase high performance liquid chromatography ( H P L C ) as previously described [33]. U n d e r these conditions, 1 0 - 2 0 % conversion of T to 5~-reduced products ( D H T and androstanedione) (h) was observed in 20 min incubations. In control studies using m o c k (no D N A ) transfected cells, up to 20% conversion of T to androstenedione from endogenous 17-keto-steroid dehydrogenase was observed, but no 5~-reduced products were produced. For inhibitor studies, finasteride was dissolved in ethanol and added to the culture to give the desired concentration. T h e a m o u n t of ethanol added was kept constant at 0.3% for type 1 and 0.2% for type 2 5~R. In vitro assays
Cell pellets containing recombinant rat types 1 or 2 5~R were resuspended in 1 M sucrose, 1 m M 3-[Nmorpholino]propanesulfonic acid (MOPS), p H 7 . 2 , 20 m M potassium chloride, 1 m M dithiothreitol ( D T T ) , 1 m M phenylmethyl sulfonyl fluoride and 5 m M N A D P H . T h e cells were broken by three freeze/thaw cycles in a dry ice/methanol bath. T h e suspension was then sonicated for 4 x 30 s intervals on ice, with 2 min between sonications. T h e suspension was stored at - 8 0 ° C . T h e reaction mixture for the rat type 1 5~R contained 33 m M succinic acid, 44 m M imidazole,
Inhibition of the Rat 5~-Reductases by Finasteride
57
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33 m M diethanolamine (SID), p H 6.5, 2 p M [3H]-T, 1 m M D T T and 0.5 m M N A D P H in a final volume of 0.1 ml. T h e assay was initiated by the addition of enzyme and incubated at 37°C for 2 0 - 3 0 min. T h e assay mixture of the rat type 2 5~R contained similar components, but in this case the SID buffer was p H 5.5 and the concentration of [ 3 H ] - T was 0.15 pM. For both isozymes, the reaction was quenched with cyclohexane:ethylacetate (70:30v/v) and T separated from D H T by normal phase H P L C . Inhibitor studies Finasteride was dissolved in 100% ethanol. T h e concentration of ethanol in the assay mixture was constant at 0.5%. ICso values were determined at K m concentrations of T (type 1, 2 pM; type 2, 0.15 pM) using a five-point titration where the concentration of the inhibitor varied from 0.1 to 1000 nM. T h e m e c h a n i s m of action of the inhibitor was determined using the reaction conditions described above with varying concentrations of inhibitor. Aliquots of the reaction were quenched at various times. Progress curves for type 1 5~R were best fit to a linear model (equation 1) with standard errors <2%. T h e calculated initial velocities (vo) were fit to equation 2 using non-linear regression to give Ki -- 1 0 . 2 _ 1.3 n M where A represents T concentration which was held constant at Km = 2 pM. T i m e - d e p e n d e n t inhibition of the rat type 2 5~R was analysed using the model originally developed by Morrison and Walsh [34]. Given that the formal kinetic mechanism of 5~R with the substrate is ordered bi bi with the co-factor adding first and leaving last [36], a similar model was used to describe the inhibition of the h u m a n 5~Rs by finasteride where E represents enzyme with nicotinamide-bound co-factor rather than free enzyme [27-29]. Ki
E + I ~
k3
E ' 1 ~--~E " I*
(Scheme 1)
k4
T h e time courses were fit by non-linear regression to an integrated first-order rate equation 3, where vo is the initial velocity, vs is the infinite-time velocity, and kob~ is the rate constant for progression of the enzyme between these two steady states. Standard errors for the determination of kob~ were <5%. T h e infinite-time velocities were set equal to zero, because the inhibition generally appeared to go to completion over the entire range of inhibitor concentrations. T h e calculated initial velocities (vo) were fit to equation 2, where A represents T concentration which was held constant at Km = 0.15/~M. N A D P H was included at 5 0 0 # M , a concentration which greatly exceeds the low /~M K m [ 3 5 ] , therefore the contribution of N A D P H can be ignored in this analysis. U n d e r these conditions, K i = 1.19 + 0.10 n M was determined for the preliminary complex of finasteride with rat type 2 57R.
T h e observed pseudo-first-order rate constants (kobs) were then fit to equation 4, using this value for Ki. This gave k 3 = 1.62 + 0.22 x 10-3/s. T h e value for k4, the rate constant for dissociation and the y-intercept, was essentially equal to the rate constant for loss of enzymatic activity observed in the control reaction in the absence of inhibitor, and should not be taken to imply that inhibition is reversible; exchange experiments (not described) indicate that the true value for k4 is virtually zero. T h e second-order rate constant for time-dependent inhibition by finasteride is thus k 3 / Ki = 1.37 _ 0.31 × 1 0 6 -/~fx/S. (1) (2) (3) (4)
y = V0t + Y0 Vo = VmaxA/(Ka(1 + (I/Ki)) + A) y = vst + (Vo - vs)(1 - e-kt)/k + Yo kobs = k 4 + k3[(I/Ki)/(1 + (A/Ka) + (I/Ki))].
Isolation of the N A D P adduct and dihydrofinasteride T h e rat type 2 isozyme-[3H]-finasteride complex was formed in a reaction containing 25 ~tl of the recombinant enzyme h o m o g e n a t e ( 1 . 4 r a g protein, specific activity 160 pmol/min/mg), 125 nmol NADPH, and 55 p m o l [3H]-finasteride (1.12 x 1 0 6 dpm, specific activity =20.273 dpm/frnol) and buffer in a total volume of 0.25 ml. T h e buffer consisted of 0.1 M M O P S , 1 m M ethylene diamine tetraacetic acid ( E D T A ) , and 0.1% bovine serum albumin (BSA) at p H 7 . 2 0 . T h e suspension was incubated for 2 h at 37°C, which is equivalent to some 50 half-lives at the saturating [3H]-finasteride concentration employed here (k3 =0.0016/s). Afterwards, the excess [3H]-finasteride was removed by dialysis overnight against 0.001 M M O P S buffer at p H 7.2 and 4°C. T h e dialysed enzyme solution contained 2 9 7 0 0 d p m tritium and represented an incorporation of 2.65% of the original [3H]-finasteride. This labelled enzyme-inhibitor complex was heat denatured to bring about the release of the expected NADPH-[3H]-dihydrofinasteride adduct and to catalyse its decomposition to [3H]-dihydrofinasteride. T o this end, the dialysed protein solution (1.33 ml) was m a d e 0.1 M in M O P S , p H 7.20, and then incubated in a boiling water bath for 30 min. T h e solution was clarified by centrifugation at 10,000g in a microfuge and then analysed by reverse-phase chromatography on a Vydac C-18 column ( 4 . 6 × 2 5 0 m m , 300.~) employing 60% aqueous methanol as the mobile phase. U n d e r these conditions, the polar adduct emerges with the flow-through peak (4 min) and dihydrofinasteride emerges at ~ 1 4 min. Although the absolute retention times of the steroids are variable, + 3 m i n , dihydrofinasteride is easily distinguished from finasteride by co-injection of finasteride as an internal standard.
Inhibition of the Rat 5~-Reductases by Finasteride
Table 1. Inhibition of 5~R isozymes by finasteHde ICS0 (nM) Rat* Crude enzyme preparations Whole cell assay Human* Crude enzyme preparations
Type 1
Type 2
]~3 _1.6
1.0+0.1
23 +_0.32
5.2 + 0.26
670
4.2
*Values represent the IC5o + SE. *Ref. [33] ..
RESULTS
Inhibition of rat 5c~Rs by finasteride in intact cells Before in vitro investigation of the m e c h a n i s m of the inhibition of rat 5,~Rs by finasteride, whole cell studies were conducted using C O S cells expressing these enzymes. In these: experiments, cells were transfected with expression plasmids containing the rat types 1 or 2 5c~R cDNAs. 2-3 days later, the m e d i u m was withdrawn and replaced with PBS containing [3H]-T. Steroid 5c~R activity was assayed by monitoring the appearance of 5~-reduced products in the buffer by H P L C . T h e concentration of finasteride required to inhibit 50% of the conversion of T to product (ICs0) by C O S cells expressing type 1 or type 2 5:~R is indicated in T a b l e 1. T h e s e studies indicate that finasteride is a potent inhibitor with ICso values of 23 + 0.32 n M and 5.2 + 0.26 n M for rat types 1 and 2 5~R, respectively. T h e time course for development of the inhibition by finasteride was followed with progress curves for the formation of 5~-reduced products in C O S cells expressing rat types 1 or 2 5~R. In these studies, as
before, the m e d i u m was replaced with buffer containing [3H]-T. At selected times, aliquots were withdrawn and analysed for 5~-reduced products by H P L C . Typical time courses are shown in Fig. 2. After a short lag phase of ~15 min, the linear production of 5or-reduced products was observed in the absence of inhibitor with either rat type 1 or 2 5~R. W h e n 20 n M finasteride was included at the time of addition of radiolabelled subs*rate, the rate of product formation was reduced with the rat type 1 isozyme (Fig. 2). Importantly, there was no evidence of a time-dependent c o m p o n e n t to the inhibition. In contrast, the inclusion of 3 n M finasteride with cells expressing the rat type 2 5ctR resulted in a timedependent drop in the formation of products (Fig. 2). By 6 0 - 7 5 rain, complete inhibition of the type 2 isozyme was observed in the presence of 3 n M finasteride. T h e time courses of inhibition shown in Fig. 2 imply differences in the m e c h a n i s m of inhibition of the rat 5~Rs by finasteride. It appears that finasteride displays reversible inhibition of rat type 1 5~R and time-dependent inhibition of rat type 2 5~R. Washout experiments were designed to demonstrate that rat type 2 5c~R is unable to recover from the inhibition by finasteride. Studies were conducted with the rat type 1 isozyme in parallel as a control. In these experiments, intact ceils expressing the isozymes of 5~R were incubated with finasteride. Product formation was monitored using [3H]-T as before. After a 60 min incubation, which was ample time for the inhibition to develop, buffer containing inhibitor was withdrawn and replaced with fresh buffer and [3H]-substrate. As indicated in Fig. 3, control incubations (minus finasteride) were linear in b o t h phases of the experiment. Importantly, the rate of product formation was similar during b o t h stages of the study. T h e incubation of
50
Type 1
59
Type 2
•
30 40 •
•
Control
30
zo Q.
o~ 20 10 10
0
0
20
40 minutes
60
80
0
I
20
40 minutes
I
60
80
Fig. 2. R e a c t i o n p r o g r e s s c u r v e s with COS cells e x p r e s s i n g t h e rat 50d~s in t h e p r e s e n c e a n d a b s e n c e o f finast e r l d e . C e l l s w e r e t r a n s f e c t e d w i t h rat type 1 (left p a n e l ) or type 2 (right panel) 5~R expression p l a s m i d s a n d a s s a y e d 48 h later with [3HI-T as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A t s e l e c t e d t i m e s afiquots w e r e withdrawn, e x t r a c t e d with an equal v o l u m e o f c y c l o h e x a n e : e t h y l a c e t a t e (55:45) a n d a n a l y s e d b y n o r m a l p h a s e H P L C . ~--o, Conl~ol; c - - o , finasteride. Results p r e s e n t e d a r e a r e p r e s e n t a t i v e e x a m p l e f r o m four i n d e p e n d e n t d e t e r m i n a t i o n s w h i c h a g r e e d within 20-25%.
60
B. A z z o l i n a et al.
15 Type 1
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. / ~
10
/
/
10
0
15 Type 2
20
wash
0
40
60 80 minutes
100
120
20
40
60 80 minutes
100
120
Fig. 3. W a s h - o u t e x p e r i m e n t s c o n d u c t e d w i t h C O S ceils t r a n s f e c t e d w i t h r a t 50dRs. I n t a c t cells e x p r e s s i n g t h e r a t t y p e 1 (left p a n e l ) a n d t y p e 2 ( r i g h t p a n e l ) 50dR w e r e a s s a y e d w i t h [ 3 H ] - T i n t h e p r e s e n c e o r a b s e n c e o f f i n a s t e r i d e a n d 5 ~ - r e d u c e d p r o d u c t s w e r e m o n i t o r e d b y H P L C . A f t e r a 60 r a i n i n c u b a t i o n , b u f f e r w a s w i t h d r a w n a n d r e p l a c e d w i t h f r e s h b u f f e r a n d [3H]-T. A l i q u o t s w e r e w i t h d r a w n at s e l e c t e d t i m e s , e x t r a c t e d a n d a n a l y s e d b y H P L C . o r e . , C o n t r o l ; o---o f i n a s t e r l d e .
20 n M finasteride with type 1 5~R resulted in an 80% decrease in the rate of product formation c o m p a r e d to the control. In the second phase of the experiment, when the buffer without inhibitor was employed, enzyme activity was restored to the control level, indicating that the inhibition by finasteride is reversible. In contrast, 3 n M finasteride resulted in time-dependent inhibition of the rat type 2 isozyme. However, enzyme activity was not recovered when finasteride was removed, thus the inhibition appears to be irreversible. In vitro inhibition of rat type 1 and 2 5~R
Studies using enzyme preparations of rat types 1 and 2 5~R also indicated that finasteride is a potent inhibitor of both isozymes with ICs0 values of 13 n M and 1 nM, respectively (Table 1). Therefore, in fixed time assays using whole cell assays or crude prep-
5
arations of rat types 1 or 2 5aR, finasteride displays only a 4-13-fold selectivity for rat type 2 5~R compared to >100-fold selectivity for h u m a n type 2 5~R (Table 1). These results are in good agreement with results reported previously [20]. Time courses for inhibition
In experiments designed to evaluate further the m e c h a n i s m of inhibition of enzyme preparations of rat types 1 or 2 5~R, progress curves of product formation were conducted in the presence and absence of finasteride. As presented in Fig. 4, linear progress curves were observed with the rat type 1 5~R in the presence of finasteride. It can be concluded that the inhibition developed rapidly with no evidence of a time-dependent component. An apparent Ki value of 1 0 . 2 + 1 . 3 n M was estimated for finasteride from this experiment. This value is in
16
Type 1
Type 2
ontrol
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1 0 0
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25 nM
2
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40 nM
10
15 20 minutes
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35
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Fig. 4. R e a c t i o n p r o g r e s s c u r v e s o f D H T f o r m a t i o n b y r a t t y p e 1 a n d t y p e 2 5~Rs w i t h f i n a s t e r i d e at t h e c o n c e n t r a t i o n s i n d i c a t e d . L e f t p a n e l : r a t e s o f p r o d u c t f o r m a t i o n b y r a t t y p e 1 50dR w e r e b e s t fit to a l i n e a r m o d e l ( e q u a t i o n 1). V e l o c i t i e s i n t h e p r e s e n c e / a b s e n c e o f f i n a s t e r i d e w e r e a n a l y s e d u s i n g e q u a t i o n 2 to give Ki = 10.2 + 1.3 n M . o, E x p e r i m e n t a l d a t a , _ _ _ t h e o r e t i c a l fine. R i g h t p a n e l : p r o g r e s s c u r v e s f o r r a t t y p e 2 50dR w e r e fit i n d i v i d u a l l y to e q u a t i o n 3 a n d a n a l y s e d a s d e s c r i b e d in M a t e r i a l s a n d m e t h o d s to give t h e s e c o n d o r d e r r a t e c o n s t a n t kflKi = 1.37 _+ 0.31 × 106/~[]s. o, E x p e r i m e n t a l d a t a ; _ _ _ , t h e o r e t i c a l fine.
Inhibition of the Rat 5ct-Reductases by Finasteride good agreement with results presented previously with rat prostate 5~R [6, 36]. Progress curves conducted with rat type 2 5~tR yielded different results. As indicated in Fig. 4, inhibition by finasteride appeared to be time dependent. A fit of these time courses to scheme 1 indicates that finasteride forms a preliminary Michaelis complex with the enzyme and N A D P H with Ki = 1.19_+ 0 . 1 0 n M , which is converted to the high-affinity complex (EI*) with a rate constant k3 = 1.62 + 0.:22 x 10-3/s. T h e second-order rate constant for development of inhibition, which applies at inhibitor concentrations below Ki, is k3/ K i = 1.37 + 0.31 x 10 6 I n / s . T h i s rate constant is comparable to that for the inhibition of the h u m a n type 2 isozyme, measured under somewhat different conditions [29].
61
A
3H
I 0
I 5
I 10
I 15
I 20
I 25
Retention Time (rain)
Finasteride
B
Evidence of N A D P + adduct formation by rat type 2 5~R Results from these laboratories indicate that finasteride is a mechanism-.based inhibitor of the h u m a n 5~Rs [29]. Accepted as an alternative substrate, it is ultimately reduced to dihydrofinasteride, but this takes place through an e n z y m e - b o u n d N A D P - d i h y drofinasteride adduct. T h e adduct is released so slowly from the enzyme that the inhibition is essentially irreversible. T h e m e c h a n i s m of inhibition and structure of this adduct are shown in Fig. 1. Studies were conducted with rat type 2 5~R and [3H]-finasteride in order to address whether the same m e c h a n i s m could explain the time-dependent inhibition of this i s o z y m e It was found that inhibition with [3H]-finasteride resulted in an enzyme-inhibitor complex that was not reversible by dialysis. H e a t denaturation of this complex produced two [3H]-products which were readily separated by H P L C [Fig. 5(A)]. T h e polar material eluting in the void volume of the column m a y represent the N A D P [3H]-dihydrofinasteride adduct previously identified with the h u m a n 5~Rs. Additional studies are necessary to confirm this assignment. T h e [3H]-product eluting at 14 min migxates with the same retention time of dihydrofinasteride. Co-injection with authentic [3H]-finasteride (retention time 12.8 min) definitively established th,'tt neither product co-migrated with untransformed inhibitor [Fig. 5(B)]. U p o n heat denaturation, the percentage conversion to dihydrofinasteride (67% in 3 0 m i n ) is consistent with that observed previously wiEh the h u m a n isozymes [29]. In contrast to these results with the rat type 2 isozyme, this m e c h a n i s m does not appear to contribute significantly to the inhibition of rat type 1 isozyme by finasteride. T h e r e is no evidence of time-dependent inhibition (Fig. 4), which argues very strongly that finasteride is a simple, competitive, rapidly reversible inhibitor of this isozyme. Moreover, there is no detectable production of [3H]-dihydrofinasteride in solutions of the enzyme, N A D P H and [3H]-finasteride (data not shown).
I
I
I
I
I
I
0
5
10
15
20
25
Retention Time (rain)
Fig. 5. C h r o m a t o g r a p h i c analysis o f 3H r e l e a s e d f r o m rat type 2 50d1 after i n a c t i v a t i o n by [3H]-finasteride. (A) E n z y m e : i n h i b l t o r c o m p l e x w a s p r e p a r e d b y i n c u b a t i n g rat type 2 5~R with [3H]-finasteride. T h e s a m p l e w a s dialysed a n d heat d e n a t u r e d for 30 m i n . A portion o f the s a m p l e w a s a n a l y s e d b y r e v e r s e - p h a s e H P L C . T h e two p r o d u c t s elute with retention t i m e s o f ~4 m i n a n d 14 rain. (B) Co-injection d e m o n s t r a t e s that a u t h e n t i c [3H]-finasteride (retention t i m e , 1 2 . 8 m i n ) does not c o - m i g r a t e with the p r o d u c t s r e l e a s e d f r o m the enzyme.
DISCUSSION
Finasteride was originally described as a reversible, competitive inhibitor with a Ki value ~ 6 n M for the 5~R in rat prostate and liver [6, 36]. At that time it was thought that there was a single form of 5c~R [4]. N o r m i n g t o n and Russell [20] definitively established that there are two genes for 5~R in the rat encoding what are now defined as type 1 and 2 5c~R. T h e presence of two isozymes in the rat warranted a re-examination of the m e c h a n i s m of inhibition of the individual isozymes by finasteride because it is unclear which isozyme was previously studied. Initial work in our laboratory was conducted with native sources (i.e. tissue extracts) of the rat 5~Rs. T h e difference in p H dependence of the isozymes was exploited to enhance the sensitivity for rat types 1 and 2 5c~R [20]. F r o m these studies it was concluded there were differences in the m e c h a n i s m of inhibition of rat types 1 and 2 5aR by finasteride; however, definitive studies required the use of recombinant 5~Rs to ensure that
62
B. Azzolina
the enzyme preparation was homogeneous with respect to isozyme content. As reported in this paper, studies using both intact cells and enzyme preparations of the isozymes confirmed that finasteride is a reversible inhibitor of rat type 1 and a time-dependent inhibitor of rat type 2 5~tR. T h e finding that finasteride is a reversible inhibitor of rat type 1 isozyme is in good agreement with the original reports concerning the m e c h a n i s m of inhibition of 5ctR by this c o m p o u n d . It is reasonable to assume that under the neutral p H conditions of those studies type 1 5~R activity predominated; therefore conclusions drawn from early studies are consistent with those reported in this p a p e r that finasteride is a reversible, competitive inhibitor. Furthermore, it is now understandable why the more complicated m e c h anism-based inhibition for the h u m a n 5~Rs did not come to light until recombinant enzymes were available. Most early kinetic studies were conducted with 5ctR from the rat liver because this tissue was readily available and contained >100-fold higher levels of enzyme activity c o m p a r e d to h u m a n tissues [8, 37]. Rat liver is enriched in rat type 1 5~R and accordingly reversible inhibition by finasteride was observed. T h e r e is good evidence that finasteride displays time-dependent inhibition of rat type 2 5~R (Figs 2 and 4). However, this observation alone provides little insight into the details of the m e c h a n i s m of inhibition. Previous studies with the h u m a n 5c~Rs relied on product identification after incubation with [3H]-finasteride to dissect the m e c h a n i s m of inhibition shown in Fig. 1 [29]. T h e chromatographic and spectroscopic properties of the material released from the h u m a n enzymes were consistent with an N A D P adduct. Furthermore, heat denaturation of the h u m a n 5~R-inhibitor complex liberated dihydrofinasteride, the structure of which was confirmed by mass spectroscopy. A similar approach was used to assess the inhibition of the rat type 2 5~R by finasteride. H e a t denaturation of the rat enzyme-[3H]-inhibitor complex produced two products which migrated by reverse-phase H P L C as expected for the N A D P adduct and dihydrofinasteride (Fig. 5). Additional characterization was not possible because of the limited amounts of rat type 2 5~R produced by transient transfection in C O S cells. However, these preliminary findings with rat type 2 5~R, in the light of the results with the baculovirus-expressed h u m a n 57Rs, suggest that mechanism-based inhibition is involved in the inactivation of rat type 2 isozyme by finasteride. T h e reversible inhibition of rat type 1 5~R by finasteride is somewhat surprising given that m e c h a n i s m based inhibition by this c o m p o u n d has now been established for rat type 2, h u m a n type 1 and h u m a n type 2 5erRs [29]. Why is finasteride unable to inactivate rat type 1 5~R irreversibly? T h e first step in the m e c h a n i s m presented in Fig. 1 requires that enzymes recognize finasteride as an alternative substrate and
et al.
catalyse reduction of the double b o n d in the A-ring of the azasteroid. I f the enzyme is incapable of this reaction, reversible inhibition by finasteride will result. Alternatively, reduction of finasteride could occur, but the partitioning of the enolate might favor proton transfer to yield dihydrofinasteride rather than collapse with N A D P ÷ to form the stable adduct (see Fig. 1). In order to distinguish between these possibilities, rat type 1 5ctR was incubated with 3H-finasteride and the product(s) analysed by H P L C . N o conversion of finasteride to dihydrofinasteride was detected. Therefore, finasteride is a reversible inhibitor of rat type 1 5~R because the enzyme is unable to catalyse the reduction of finasteride, the first step required for mechanism-based inhibition. It is worth noting that rat and h u m a n type 1 5~Rs appear to be more closely related (60% sequence identity) than are the h u m a n type 1 and type 2 isozymes (50%). However, despite the lower sequence homology of the h u m a n isozymes and the p o o r inhibition of h u m a n type 1 5c~R by finasteride (Table 1), mechanism-based inhibition of b o t h h u m a n homologs is observed. T h e distinct m e c h a n i s m of inhibition found with rat type 1 5c~R suggests that a subtle difference exists in the N A D P H - b i n d i n g site of this isozyme which interferes with enzyme-catalysed reduction of A1-4-azasteroids but not A4-3-keto-steroids (e.g. T). As noted previously, 5~R does not contain a consensus N A D P H binding site [38]. Furthermore, this class of enzymes has been intractable to purification and consequently no structural information is available. However, structure-function information was inferred from characterization of mutations in the h u m a n type 2 gene from subjects with the inherited 5ctR defciency [38]. T w o amino acids were proposed to contribute to the T binding site; both of which are conserved a m o n g isozymes and species. Eight amino acids were tentatively identified as contributing to the N A D P H - b i n d i n g site. Strict conservation is observed in seven of these residues when protein sequences of types 1 and 2 5~R from rat, cynomolgus and h u m a n are c o m p a r e d [19-21, 39]. Interestingly, arginine at position 145 of the h u m a n type 2 protein is conserved in all isozymes with the exception of rat type 1 where cysteine is found at the corresponding residue. Whether substitution at this position contributes to the different m e c h a n i s m of inhibition by finasteride remains to be determined. CONCLUSION Finasteride is a potent inhibitor of both types 1 and 2 5~R in rat. As described in this paper, species differences in the inhibition by finasteride are seen in both potency and in the m e c h a n i s m of the inhibition. Finasteride is a mechanism-based inhibitor of the h u m a n types 1 and 2 5c~R with >100-fold selectivity for h u m a n type 2 [29]. Surprisingly, finasteride is a
Inhibition of the Rat 5ct-Reductases by Finasteride
potent, reversible inhibitor of the rat type 1 57R (IC50 13 nM) but a m e c h a n i s m - b a s e d inhibitor of the rat type 2 isozyme (ICso 1 nM). In the light of these findings, one needs to consi~der whether the biological activity of finasteride in tl~e various in vivo rat models is caused by the inhibition of b o t h isozymes of 5~R.
REFERENCES 1. Wilson J. D., Griffin J. E. and Russell D. W.: Steroid 5~reductase 2 deficiency. Endocr. Rev. 14 (1993) 577-593. 2. Imperato-McGinley J., Guerrero L., Gautier T. and Peterson R. E.: Steroid 5~-reductase deficiency in man: an inherited form of male pseudohernmphroditism. Science 27 (1974) 12131215. 3. Peterson R. E., Imperato-McGinley J., Gautier T. and Sturla E.: Male pseudohermaphroditism due to steroid 5~-reductase deficiency. Am, .7. Med. 62 (1977) 170-191. 4. Imperato-McGinley J. and Gautier T.: Inherited 5~-reductase deficiency in man. Trends Genet. 2 (1986) 130-133. 5. Imperato-McGinley J., Gautier T., Cai L. Q., Yee B., Epstein J. and Pochi P.: The ardrogen control of sebum production. Studies of subjects with dihydrotestosterone deficiency and complete androgen insensitivity..7. Clin. Endocr. Metab. 76 (1993) 524-528. 6, Liang T., Cascieri M~., Cheung A., Reynolds G. and Rasmusson G.: Species differences in prostatic steroid 5~reductases of rat, human and dog. Endocrinology 117 (1985) 571-579. 7. Gormley G. J., Stoner E., Brnskewitz R. C., ImperatoMcGinley J., Walsh P. C., McConnell J. D., Andriole G. L., Geller J., Bracken B., Tenover J. S., Baughan E. D., Pappas F., Taylor A., Binkowitz B., Ng J. and Group F. S.: The effect of finasteride in men with benign prostatic hyperplasia. N. Engl. J. Med. 327 (1992) 1185-1191. 8. Levy M. A., Brandt M., Heys R., Holt D. A. and Metcalf B. W.: Inhibition of rat liver steroid 5~-reductase by 3androstene-3-carboxylic acids: mechanism of enzyme inhibitor interaction. Biochemistry 29 (1990) 2815-2824. 9. Audet P., Nurcombe H., Lamb Y., Jorasky D., Lloyd-Davis K. and Morris: Effect of multiple doses of epristeride (E) a steroid 5~-reductase inh!ibitor on serum dihydrotestosterone in older male subjects. Clin Pharmacol. Ther. 53 (1993) 231. 10. Hirosumi J., Nakayama O., Fagan R., Sawada K., Chida N., Inami M., Takahashi S.: Kojo H., Notsu Y. and Okuhara M.: FK143, a novel nonsteraidal inhibitor of steroid 5ct-reductase: (1) In vitro effects on human and animal prostatic enzymes. .7. Steroid Biochem. Molec. Biol. 52 (1995) 357-363. 11. di Salle E., Guidici D., Briatico G., Ornati G. and Panzeri A.: Hormonal effects of turosteride, a 5-alpha-reductase inhibitor, in the rat..7. Steroid Biochem. Molec. Biol. 46 (1993) 549-555. 12. Frye S. V., Haffner C. D., Maloney P. R., Mook R. A. Jr, Dorsey G. F., Hiner R. N., Batchelor K. W., Bramson H. N , Stuart J. D., Schweiker S. L., van Arnold J., Bickett D. M., Moss M. L., Tian G., Unwalla R. J., Lee F. W., Tippin T. K., James M. K., Grizzle M. K., Long J. E. and Schuster S. V.: 6-azasteroids: Potent dual inhibitors of human type 1 and 2 steroid 5~-reductase..7. Med. Chem. 36 (1993) 4313-4315. 13. Frye S. V. et al.: 6-Aza:~teroids: structure-activity relationships for inhibition of type 1 and 2 5~-reductase and human adrenal 3fl-hydroxy-AS-steroid dehydrogenase/3-keto-AS-isomerase, J. Med. Chem. 37 (19941) 2352-2360. 14. Brooks J. R., Berman C., Primka R. L., Reynolds G. F. and Rasmusson G. H.: Five-alpha-reductase inhibitory and antiandrogenic activities of so:me 4-azasteroids in the rat. Steroids 47 (1986) 1-19. 15. Clark R. L., AntoneUo J. M., Grossman S. J., Wise L. D., Anderson C., Bagdon W. J., Prahalada S., MacDonald J. S. Robertson R. T.: External genitalia abnormalities in male rats exposed in utero to fmasteride, a 5ct-reductase inhibitor. Teratology 42 (1990) 91--100. 16. Clark R. L., Anderson C. A., Prahalada S., Robertson R. T., Lochry E. A., Leonard Y. M., Stevens J. L. and Hoverman A. M.: Critical developmerLtal periods for effects on male rat geni-
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talia induced by fmasteride, a 5~-reductase inhibitor. Toxicol. AlyPL Pharrnacol. 119 (1993) 34-40. Imperato-McGinley J., Binienda Z., Arthur A., Mininberg D. T., Vaugh J. E.D. and Quimby F. W.: The development of a male pseudoherrnaphroditic rat using an inhibitor of the enzyme 5~-reductase. Endocrinology 116 (1985) 807-812. Andersson S., Bishop R. W. and Russell D. W.: Expression cloning and regulation of steroid 5~-reductase, an enzyme essential for male sexual differentiation, ft. Biol. Chem. 12 (1989) 16249-16255. Andersson S., Berman D. M., Jenkins E. P. and Russell D. W.: Deletion of steroid 5~-reductase 2 gene in male pseudohermaphroditism. Nature 354 (1991) 159-161. Normington K. and Russell D. W.: Tissue distribution of kinetic characteristics of rat steroid 5~-reductase isozymes..7. Biol. Chem. 267 (1992) 19548-19554. Levy M. A., Brandt M., Sheedy K. M., Holt D. A., Heaslip J. I., TriU J. J., Ryan P. J., Morris R. A., Garrison L. M. and Bergsma D. J.: Cloning, expression and functional characterization of type 1 and type 2 steroid 5-alpha-reductases from cynomolgus monkey: comparisons with human and rat isozymes..7. Steroid Biochem. Molec. Biol. 54 (1995) 307-319. Thigpen A. E., Silver R. I., Guileyardo J. M., Casey M. L., McConneU J. D. and RusseU D. W.: Tissue distribution and ontogeny of steroid 5~-reductase isozyme expression..7. Clin. Invest. 92 (1993) 903-910. Levy M., Metcalf B. W., Brandt M., Erb J. M., Oh H.-J., Geaslip J. I., Yen H.-K., Rozamus L. W. and Holt D. A.: 3Phosphinic acid and 3-phosphonic acid steroids as inhibitors of steroid 5~-reductase: species comparison and mechanistic studies. Bioorg. Chem. 19 (1991) 245-260. Holt C. A., Levy M. A., Yen H.-W., Oh H.-J. and Metcalf B. W.: Inhibition of steroid 5~-reductase by 3-nitrosteroids: synthesis, mechanism, of inhibition, and in vivo activity. Bioorg. Med. Chem. Lett. 1 (1991) 27-32. Rhodes L., Harper J., Uno H., Gaito G., Audettearrnda J., Kurata S., Berman C., Primka R. and Pikounis B.: The effects of finasteride (Proscar) on hair growth, hair cycle stage, and serum testosterone and dihydrotestosterone in adult male and female stumptial macaques (Macaca arcotoides). .7. Clin. Endocr. Metab. 79 (1994) 991-996. Rittmaster R. S., Uno J., Povar M. L., Mellin T. N. and Loriaux D. L.: The effects of n,n-diethyl-4-methyl-3-oxo-4aza-5-alpha-androstane-17-beta-carboxamide a 5-alpha reductase inhibitor and antiandrogen on the development of baldness in the stumptail macaque..7. Clin. Endocr. Metab. 65 (1987) 188-193. Failer B., Farley D. and Nick H.: Finasteride: a slow-binding 5ct-reductase inhibitor. Biochemistry. 32 (1993) 5705-5710. Tian G., Stuart J. D., Moss M. L., Domanico P. L., Bramson H. N., Patel I. R., Kadwell S. H., Overton L. K., Kost T. A., Mook R. A. Jr, Frye S. V., Batchelor K. W. and Wiseman J. S.: 17fl-(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. Biochemistry 33 (1994) 2291-2296. Bull H. G., Garcia-Calvo M., Andersson S. A., Baginsky W. F., Chan H. K., Ellsworth D., Miller R., Stearns R. A., Bakshi R. K., Rasmusson G. H., Tolman R. L., Myers R. W., Kozarich J. W. and Harris G. S.: Mechanism-based inhibition of human steroid 5a-reductase by finasteride: enzyme catalyzed formation of NADP-dihydrofinasteride, a potent bisubstrate analog inhibitor..7. Am. Chem. Soc. 118 (1996) 2359-2365. Liang T., Heiss C. E., Ostrove S., Rasmusson G. H. and Cheung A.: Binding of a 4-methyl-4-azasteroid to 5~-reductase of rat liver and prostate microsomes. Endocrinology 112 (1983) 1460-1468. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent..7. Biol. Chem. 193 (1951) 265-275. Andersson A., Davis D. L., Dahlb~ick H., J6rnvall H. and Russell D. W.: Cloning, structure and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme..7. Biol. Chem. 264 (1989) 8222-8229. 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
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5~-reductase in human scalp. Proc. Natl. Acad. Sci. U.S.A. 89 (1992) 10787-10791. 34. 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. 35. Thigpen A. E., Cala K. M. and Russell D. W.: Characterization of chinese hamster ovary cell lines expressing human steroid 5~-reductase isozymes. J. Biol. Chem. 268 (1993) 17404-17412. 36. Levy M. A.~ Brandt J. and Greway A. T.: Mechanistic studies with solubilized rat liver steroid 5~-reductase: elucidation of the kinetic mechanism. Biochemistry 29 (1990) 2808-2815.
37. Ichihara K. and Tanaka C.: Some properties of progesterone 5~-reductase solubilized from rat liver microsomes. Biochem. Int. 15 (1987) 1005-1011. 38. Wigley W. C., Prihoda J. S., Mowszowicz I., Mendonca B. B., New M. I., Wilson J. D. and Russell D. W.: Natural mutagenesis study of the human steroid 5c~-reductase 2 isozyme. Biochemistry 33 (1994) 1265-1270. 39. Andersson S. and Russell D. W.: Structural and biochemical properties of cloned and expressed human and rat steroid 5~reductases. Proc. Natl. Acad. Sci. U.S.A. 87 (1990) 36403644.
Pergamon
I n h i b i t i o n of Rat -Reductases by Finasteride: E v i d e n c e for Isozyrne D i f f e r e n c e s in the M e c h a n i s m o f Inhibition B. A z z o l i n a , ' K. E I I s w o r t h , 1 S . A n d e r s s o n , 2 W. G e i s s l e r , ' H . G. Bull' a n d G. S. H a r r i s 1. IDepartment of Enzymoi!ogy, Merck Research Laboratories, PO Box 2000, R80Y-140, Rahway, N J 07065, U.S.A. and 2Department of Obsi~,etrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, T X 75235, U.S.A.
T h e m e c h a n i s m o f ird~ibition o f t h e r a t t y p e s 1 a n d 2 5 ~ - r e d u c t a s e b y f i n a s t e r i d e w a s i n v e s t i g a t e d u s i n g r e c o m b i n a n t l y e x p r e s s e d e n z y m e s . T h e s e s t u d i e s r e v e a l e d t h a t f i n a s t e r i d e is a p o t e n t , r e v e r s ible i n h i b i t o r o f t h e rat t y p e 1 5 ~ - r e d u c t a s e w i t h Ki = 10.2 _+ 1.3 n M . F i n a s t e r i d e is a p o t e n t i n h i b i t o r o f t h e r a t t y p e 2; h o w e v e r , in t h i s c a s e t h e c o m p o u n d b i n d s to t h e t y p e 2 i s o z y m e - N A D P H complex to f o r m a t e r n a r y c o m p l e x w i t h Ki = 1.19 + 0.10 n M , w h i c h t h e n r e a r r a n g e s to a h i g h a f f i n i t y c o m p l e x (E:I*) w i t h a p s e u d o first o r d e r rate c o n s t a n t o f 1.62 + 0.22 × 10-3Is. T h e s e c o n d o r d e r r a t e c o n s t a n t is kflKi = 1.37 + 0.31 × 10 6 MJs. H e a t d e n a t u r a t i o n o f t h e ( t y p e 2 e n z y m e : i n h i b i t o r ) c o m p l e x r e l e a s e s d i h y d r o f i n a s t e r i d e a n d p r e s u m a b l y t h e N A D P + - a d d u c t p r e v i o u s l y i d e n t i f i e d w i t h t h e h u m a n 5~r e d u c t a s e s . T h e e f f e c t s o f f i n a s t e r i d e w e r e a l s o s t u d i e d in i n t a c t C O S ceils t r a n s i e n t l y e x p r e s s i n g t h e rat t y p e s 1 a n d 2 5 ~ - r e d u c t a s e . R e s u l t s w i t h w h o l e cell a s s a y s c o n f i r m d i f f e r e n c e s in m e c h a n i s m o f i n h i b i t i o n o f r a t t y p e s 1 a n d 2 5 ~ - r e d u c t a s e b y f i n a s t e r i d e . © 1997 E l s e v i e r S c i e n c e Ltd
J. Steroid Biochem. Molec. Biol., Vol. 61, No. 1/2, pp. 55-64, 1997
INTRODUCTION
useful inhibitors for the treatment of benign prostatic hyperplasia and male pattern baldness began shortly after the discovery of 5~R. Beginning in the 1980s, many inhibitors of 5~R were described as potential clinical candidates. These compounds include finasteride (17fl-(N-tert-butylcarbamoyl)-4-aza-5~-androst-l-en-3-one, M K 9 0 6 ) [6,7], epristeride [8, 9], FK143 [10], turosteride [11], and the 6-azasteroids [12, 13]. Most inhibitors were discovered by evaluating the in vitro activity of compounds on the 5~R activity in extracts of human prostate. Subsequent in vivo studies were often conducted in a castrate rat model to evaluate the ability of compounds to block the T-stimulated growth of the prostate. T h e predictive clinical value of this rat model depended on the presumed similarity of the h u m a n and the rat forms of 5~R. Finasteride has been used as a prototype 5~R inhibitor by many investigators to define the effects of the inhibition of this enzyme in various rat models. Brooks et al. [14] demonstrated that this inhibitor prevented T - but not D H T - s t i m u l a t e d growth of the prostate in castrated male rats. These results confirm
Steroid 5~-reductase (5~R) is the N A D P H - d e p e n d e n t enzyme required for the conversion of testosterone (T) to dihydrotestosterone ( D H T ) . This enzyme is found in androgen target tissues such as the prostate, seminal vesicle and skin, which preferentially respond to D H T rather than to T [1]. T h e production of D H T is required for the normal development of the external genitalia of tile male fetus, thus defects in this enzyme give rise to a form o f male pseudohermaphroditism where affected males have ambiguous genitalia at birth [2, 3]. Post puberty, the consequences of life-long decreases in circulating D H T are an underdeveloped prostate and little or no male pattern baldness or acne [4, 5]. These findings led to the suggestion that D H T is the androgen which supports growth of the prostate and leads to scalp hair recession [4]. As a result, the search for clinically *Correspondence to Georgianna Harris. Received 13 Aug. 1996; accepted 26 Nov. 1996. Abbreviations: 5~R, 5~-reductase; T, testosterone; D H T , drotestosterone; PBS, phosphate buffered saline
dihy55
56
B. Azzolina et al.
that finasteride does not interact with the androgen receptor but blocks the formation of D H T to inhibit the growth of the prostate. Finasteride has also been used to define the effects and critical period in gestation during which 5ctR inhibition disrupts the normal development of male rats due to the need for D H T in the differentiation of the prostate and external genitalia [15-17]. Extrapolation of these results to h u m a n male development suggests that this critical period for the action of D H T is weeks 8 - 1 2 of gestation [15, 16]. T h e continued use of rat models to support the development of 5aR inhibitors is now complicated by the identification of two genes encoding two types of 5~R in the rat, m o n k e y and h u m a n [18-21]. Despite sequence homology between the rat and h u m a n isozymes, the tissue distribution and inhibitor sensitivity varies considerably between these two species [6, 20, 22, 23]. As a result of these issues, some investigators have pursued primate models to 5~R inhibitors [21, 24-26]. Given the ongoing use of finasteride as a 5~R inhibitor in various rat models, a thorough understanding of the m e c h a n i s m of inhibition of rat types 1 and 2 5~R is required to interpret the results of these in vivo studies fully. Recently, slow-binding inhibition of h u m a n types 1 and 2 5~R by finasteride was described [27, 28]. It is now clear that the mechanism of inhibition of the 5~Rs involves reduction of the Aring of the azasteroid and subsequent adduct formation with N A D P + (Fig. 1) [29]. T o date, little attention has been devoted to studying the inhibition of the rat 5~Rs. In this manuscript we describe the mechanism of inhibition of the rat isozymes of 5~R by finasteride. Furthermore, we describe the utility of intact C O S cells expressing 5~R as an alternative to crude enzyme preparations for the mechanistic study of inhibitors of this enzyme. MATERIALS AND METHODS
[7-3H]-Testosterone was purchased from N e w England Nuclear (Boston, MA, U.S.A.). Finasteride (was provided by D r Gary Rasmusson. [1,2-3H] Finasteride was prepared by D r Avery Rosegay, M e r c k Research Laboratories, by reduction of A 1 of unlabelled c o m p o u n d with tritium gas followed by reoxidation to reintroduce the double b o n d into the A ring [30]. All chemicals were obtained from Sigma Chemical Co (St Louis, M O , U.S.A.). Protein concentrations were estimated using the m e t h o d developed by Lowry et al. [31]. Expression of 5~R
R e c o m b i n a n t plasmid expressing the rat type 1 5~R was a generous gift from D r David Russell. Rat type 2 c D N A was cloned by reverse-transcriptase-polymerase chain reaction ( R T - P C R ) . Total R N A (Promega,
Madison, WI, U.S.A.; total isolation kit) was prepared from epididymis dissected from 2-month-old (sexually mature) S p r a g u e - D a w l e y rats. After reverse transcription, type 2 5aR was P C R amplified using specific primers corresponding to bases 3 8 - 5 7 in the 5' end and 8 5 4 - 8 7 2 in the 3' end of the c D N A [20]. T h e primers were designed so that the amplification product would contain the entire coding region, flanked by B a m H 1 and H i n d I I I sites. T h e P C R product of 856 base pairs was cloned into the m a m m a lian expression vector p C M V 6 [32] using the B a m H 1 and H i n d I I I sites. Double-stranded D N A sequencing confirmed the identity of the rat type 2 5~R cDNA. C O S cells were grown in Dulbecco's m i n i m u m essential m e d i u m ( D M E M ) (Gibco BRL, G r a n d Island, NY, U.S.A.) supplemented with 10% fetal bovine serum (Hyclone, Logan, U T , U.S.A.) and transfected on day 0 by electroporation using 20 #g plasmid D N A and 10 7 cells per cuvette. Whole cell assays
C O S cells were grown and transfected as described. Control cells were m o c k transfected and handled under the same conditions. On day 2-3, the m e d i u m was removed, replaced with phosphate buffered saline (PBS) containing 1 0 0 n M [3H]-T for type 1 and 50 n M for type 2 5~R. Samples of buffer were withdrawn at various times and the steroids extracted with a mixture of cyclohexane:ethylacetate (55:45v/v). Separation of T and 5~-reduced products was accomplished by normal phase high performance liquid chromatography ( H P L C ) as previously described [33]. U n d e r these conditions, 1 0 - 2 0 % conversion of T to 5~-reduced products ( D H T and androstanedione) (h) was observed in 20 min incubations. In control studies using m o c k (no D N A ) transfected cells, up to 20% conversion of T to androstenedione from endogenous 17-keto-steroid dehydrogenase was observed, but no 5~-reduced products were produced. For inhibitor studies, finasteride was dissolved in ethanol and added to the culture to give the desired concentration. T h e a m o u n t of ethanol added was kept constant at 0.3% for type 1 and 0.2% for type 2 5~R. In vitro assays
Cell pellets containing recombinant rat types 1 or 2 5~R were resuspended in 1 M sucrose, 1 m M 3-[Nmorpholino]propanesulfonic acid (MOPS), p H 7 . 2 , 20 m M potassium chloride, 1 m M dithiothreitol ( D T T ) , 1 m M phenylmethyl sulfonyl fluoride and 5 m M N A D P H . T h e cells were broken by three freeze/thaw cycles in a dry ice/methanol bath. T h e suspension was then sonicated for 4 x 30 s intervals on ice, with 2 min between sonications. T h e suspension was stored at - 8 0 ° C . T h e reaction mixture for the rat type 1 5~R contained 33 m M succinic acid, 44 m M imidazole,
Inhibition of the Rat 5~-Reductases by Finasteride
57
0
o
o
I 13~ ~lk rn
S~ ,"
0 eo o," fy z'r"
IX
I1. IX.
.~
o
~v 0+
°~ Z-t-
e~
O
,z*d
58
B. Azzolina et al.
33 m M diethanolamine (SID), p H 6.5, 2 p M [3H]-T, 1 m M D T T and 0.5 m M N A D P H in a final volume of 0.1 ml. T h e assay was initiated by the addition of enzyme and incubated at 37°C for 2 0 - 3 0 min. T h e assay mixture of the rat type 2 5~R contained similar components, but in this case the SID buffer was p H 5.5 and the concentration of [ 3 H ] - T was 0.15 pM. For both isozymes, the reaction was quenched with cyclohexane:ethylacetate (70:30v/v) and T separated from D H T by normal phase H P L C . Inhibitor studies Finasteride was dissolved in 100% ethanol. T h e concentration of ethanol in the assay mixture was constant at 0.5%. ICso values were determined at K m concentrations of T (type 1, 2 pM; type 2, 0.15 pM) using a five-point titration where the concentration of the inhibitor varied from 0.1 to 1000 nM. T h e m e c h a n i s m of action of the inhibitor was determined using the reaction conditions described above with varying concentrations of inhibitor. Aliquots of the reaction were quenched at various times. Progress curves for type 1 5~R were best fit to a linear model (equation 1) with standard errors <2%. T h e calculated initial velocities (vo) were fit to equation 2 using non-linear regression to give Ki -- 1 0 . 2 _ 1.3 n M where A represents T concentration which was held constant at Km = 2 pM. T i m e - d e p e n d e n t inhibition of the rat type 2 5~R was analysed using the model originally developed by Morrison and Walsh [34]. Given that the formal kinetic mechanism of 5~R with the substrate is ordered bi bi with the co-factor adding first and leaving last [36], a similar model was used to describe the inhibition of the h u m a n 5~Rs by finasteride where E represents enzyme with nicotinamide-bound co-factor rather than free enzyme [27-29]. Ki
E + I ~
k3
E ' 1 ~--~E " I*
(Scheme 1)
k4
T h e time courses were fit by non-linear regression to an integrated first-order rate equation 3, where vo is the initial velocity, vs is the infinite-time velocity, and kob~ is the rate constant for progression of the enzyme between these two steady states. Standard errors for the determination of kob~ were <5%. T h e infinite-time velocities were set equal to zero, because the inhibition generally appeared to go to completion over the entire range of inhibitor concentrations. T h e calculated initial velocities (vo) were fit to equation 2, where A represents T concentration which was held constant at Km = 0.15/~M. N A D P H was included at 5 0 0 # M , a concentration which greatly exceeds the low /~M K m [ 3 5 ] , therefore the contribution of N A D P H can be ignored in this analysis. U n d e r these conditions, K i = 1.19 + 0.10 n M was determined for the preliminary complex of finasteride with rat type 2 57R.
T h e observed pseudo-first-order rate constants (kobs) were then fit to equation 4, using this value for Ki. This gave k 3 = 1.62 + 0.22 x 10-3/s. T h e value for k4, the rate constant for dissociation and the y-intercept, was essentially equal to the rate constant for loss of enzymatic activity observed in the control reaction in the absence of inhibitor, and should not be taken to imply that inhibition is reversible; exchange experiments (not described) indicate that the true value for k4 is virtually zero. T h e second-order rate constant for time-dependent inhibition by finasteride is thus k 3 / Ki = 1.37 _ 0.31 × 1 0 6 -/~fx/S. (1) (2) (3) (4)
y = V0t + Y0 Vo = VmaxA/(Ka(1 + (I/Ki)) + A) y = vst + (Vo - vs)(1 - e-kt)/k + Yo kobs = k 4 + k3[(I/Ki)/(1 + (A/Ka) + (I/Ki))].
Isolation of the N A D P adduct and dihydrofinasteride T h e rat type 2 isozyme-[3H]-finasteride complex was formed in a reaction containing 25 ~tl of the recombinant enzyme h o m o g e n a t e ( 1 . 4 r a g protein, specific activity 160 pmol/min/mg), 125 nmol NADPH, and 55 p m o l [3H]-finasteride (1.12 x 1 0 6 dpm, specific activity =20.273 dpm/frnol) and buffer in a total volume of 0.25 ml. T h e buffer consisted of 0.1 M M O P S , 1 m M ethylene diamine tetraacetic acid ( E D T A ) , and 0.1% bovine serum albumin (BSA) at p H 7 . 2 0 . T h e suspension was incubated for 2 h at 37°C, which is equivalent to some 50 half-lives at the saturating [3H]-finasteride concentration employed here (k3 =0.0016/s). Afterwards, the excess [3H]-finasteride was removed by dialysis overnight against 0.001 M M O P S buffer at p H 7.2 and 4°C. T h e dialysed enzyme solution contained 2 9 7 0 0 d p m tritium and represented an incorporation of 2.65% of the original [3H]-finasteride. This labelled enzyme-inhibitor complex was heat denatured to bring about the release of the expected NADPH-[3H]-dihydrofinasteride adduct and to catalyse its decomposition to [3H]-dihydrofinasteride. T o this end, the dialysed protein solution (1.33 ml) was m a d e 0.1 M in M O P S , p H 7.20, and then incubated in a boiling water bath for 30 min. T h e solution was clarified by centrifugation at 10,000g in a microfuge and then analysed by reverse-phase chromatography on a Vydac C-18 column ( 4 . 6 × 2 5 0 m m , 300.~) employing 60% aqueous methanol as the mobile phase. U n d e r these conditions, the polar adduct emerges with the flow-through peak (4 min) and dihydrofinasteride emerges at ~ 1 4 min. Although the absolute retention times of the steroids are variable, + 3 m i n , dihydrofinasteride is easily distinguished from finasteride by co-injection of finasteride as an internal standard.
Inhibition of the Rat 5~-Reductases by Finasteride
Table 1. Inhibition of 5~R isozymes by finasteHde ICS0 (nM) Rat* Crude enzyme preparations Whole cell assay Human* Crude enzyme preparations
Type 1
Type 2
]~3 _1.6
1.0+0.1
23 +_0.32
5.2 + 0.26
670
4.2
*Values represent the IC5o + SE. *Ref. [33] ..
RESULTS
Inhibition of rat 5c~Rs by finasteride in intact cells Before in vitro investigation of the m e c h a n i s m of the inhibition of rat 5,~Rs by finasteride, whole cell studies were conducted using C O S cells expressing these enzymes. In these: experiments, cells were transfected with expression plasmids containing the rat types 1 or 2 5c~R cDNAs. 2-3 days later, the m e d i u m was withdrawn and replaced with PBS containing [3H]-T. Steroid 5c~R activity was assayed by monitoring the appearance of 5~-reduced products in the buffer by H P L C . T h e concentration of finasteride required to inhibit 50% of the conversion of T to product (ICs0) by C O S cells expressing type 1 or type 2 5:~R is indicated in T a b l e 1. T h e s e studies indicate that finasteride is a potent inhibitor with ICso values of 23 + 0.32 n M and 5.2 + 0.26 n M for rat types 1 and 2 5~R, respectively. T h e time course for development of the inhibition by finasteride was followed with progress curves for the formation of 5~-reduced products in C O S cells expressing rat types 1 or 2 5~R. In these studies, as
before, the m e d i u m was replaced with buffer containing [3H]-T. At selected times, aliquots were withdrawn and analysed for 5~-reduced products by H P L C . Typical time courses are shown in Fig. 2. After a short lag phase of ~15 min, the linear production of 5or-reduced products was observed in the absence of inhibitor with either rat type 1 or 2 5~R. W h e n 20 n M finasteride was included at the time of addition of radiolabelled subs*rate, the rate of product formation was reduced with the rat type 1 isozyme (Fig. 2). Importantly, there was no evidence of a time-dependent c o m p o n e n t to the inhibition. In contrast, the inclusion of 3 n M finasteride with cells expressing the rat type 2 5ctR resulted in a timedependent drop in the formation of products (Fig. 2). By 6 0 - 7 5 rain, complete inhibition of the type 2 isozyme was observed in the presence of 3 n M finasteride. T h e time courses of inhibition shown in Fig. 2 imply differences in the m e c h a n i s m of inhibition of the rat 5~Rs by finasteride. It appears that finasteride displays reversible inhibition of rat type 1 5~R and time-dependent inhibition of rat type 2 5~R. Washout experiments were designed to demonstrate that rat type 2 5c~R is unable to recover from the inhibition by finasteride. Studies were conducted with the rat type 1 isozyme in parallel as a control. In these experiments, intact ceils expressing the isozymes of 5~R were incubated with finasteride. Product formation was monitored using [3H]-T as before. After a 60 min incubation, which was ample time for the inhibition to develop, buffer containing inhibitor was withdrawn and replaced with fresh buffer and [3H]-substrate. As indicated in Fig. 3, control incubations (minus finasteride) were linear in b o t h phases of the experiment. Importantly, the rate of product formation was similar during b o t h stages of the study. T h e incubation of
50
Type 1
59
Type 2
•
30 40 •
•
Control
30
zo Q.
o~ 20 10 10
0
0
20
40 minutes
60
80
0
I
20
40 minutes
I
60
80
Fig. 2. R e a c t i o n p r o g r e s s c u r v e s with COS cells e x p r e s s i n g t h e rat 50d~s in t h e p r e s e n c e a n d a b s e n c e o f finast e r l d e . C e l l s w e r e t r a n s f e c t e d w i t h rat type 1 (left p a n e l ) or type 2 (right panel) 5~R expression p l a s m i d s a n d a s s a y e d 48 h later with [3HI-T as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A t s e l e c t e d t i m e s afiquots w e r e withdrawn, e x t r a c t e d with an equal v o l u m e o f c y c l o h e x a n e : e t h y l a c e t a t e (55:45) a n d a n a l y s e d b y n o r m a l p h a s e H P L C . ~--o, Conl~ol; c - - o , finasteride. Results p r e s e n t e d a r e a r e p r e s e n t a t i v e e x a m p l e f r o m four i n d e p e n d e n t d e t e r m i n a t i o n s w h i c h a g r e e d within 20-25%.
60
B. A z z o l i n a et al.
15 Type 1
e/~
wash
"O O
o O..
•
0
. / ~
10
/
/
10
0
15 Type 2
20
wash
0
40
60 80 minutes
100
120
20
40
60 80 minutes
100
120
Fig. 3. W a s h - o u t e x p e r i m e n t s c o n d u c t e d w i t h C O S ceils t r a n s f e c t e d w i t h r a t 50dRs. I n t a c t cells e x p r e s s i n g t h e r a t t y p e 1 (left p a n e l ) a n d t y p e 2 ( r i g h t p a n e l ) 50dR w e r e a s s a y e d w i t h [ 3 H ] - T i n t h e p r e s e n c e o r a b s e n c e o f f i n a s t e r i d e a n d 5 ~ - r e d u c e d p r o d u c t s w e r e m o n i t o r e d b y H P L C . A f t e r a 60 r a i n i n c u b a t i o n , b u f f e r w a s w i t h d r a w n a n d r e p l a c e d w i t h f r e s h b u f f e r a n d [3H]-T. A l i q u o t s w e r e w i t h d r a w n at s e l e c t e d t i m e s , e x t r a c t e d a n d a n a l y s e d b y H P L C . o r e . , C o n t r o l ; o---o f i n a s t e r l d e .
20 n M finasteride with type 1 5~R resulted in an 80% decrease in the rate of product formation c o m p a r e d to the control. In the second phase of the experiment, when the buffer without inhibitor was employed, enzyme activity was restored to the control level, indicating that the inhibition by finasteride is reversible. In contrast, 3 n M finasteride resulted in time-dependent inhibition of the rat type 2 isozyme. However, enzyme activity was not recovered when finasteride was removed, thus the inhibition appears to be irreversible. In vitro inhibition of rat type 1 and 2 5~R
Studies using enzyme preparations of rat types 1 and 2 5~R also indicated that finasteride is a potent inhibitor of both isozymes with ICs0 values of 13 n M and 1 nM, respectively (Table 1). Therefore, in fixed time assays using whole cell assays or crude prep-
5
arations of rat types 1 or 2 5aR, finasteride displays only a 4-13-fold selectivity for rat type 2 5~R compared to >100-fold selectivity for h u m a n type 2 5~R (Table 1). These results are in good agreement with results reported previously [20]. Time courses for inhibition
In experiments designed to evaluate further the m e c h a n i s m of inhibition of enzyme preparations of rat types 1 or 2 5~R, progress curves of product formation were conducted in the presence and absence of finasteride. As presented in Fig. 4, linear progress curves were observed with the rat type 1 5~R in the presence of finasteride. It can be concluded that the inhibition developed rapidly with no evidence of a time-dependent component. An apparent Ki value of 1 0 . 2 + 1 . 3 n M was estimated for finasteride from this experiment. This value is in
16
Type 1
Type 2
ontrol
4
12 y
~
10 nM
3
i
tr°l
"o o
o 13.
#_
•
1 0 0
nM
25 nM
2
5
40 nM
10
15 20 minutes
25
30
35
10
20
30
40
50
minutes
Fig. 4. R e a c t i o n p r o g r e s s c u r v e s o f D H T f o r m a t i o n b y r a t t y p e 1 a n d t y p e 2 5~Rs w i t h f i n a s t e r i d e at t h e c o n c e n t r a t i o n s i n d i c a t e d . L e f t p a n e l : r a t e s o f p r o d u c t f o r m a t i o n b y r a t t y p e 1 50dR w e r e b e s t fit to a l i n e a r m o d e l ( e q u a t i o n 1). V e l o c i t i e s i n t h e p r e s e n c e / a b s e n c e o f f i n a s t e r i d e w e r e a n a l y s e d u s i n g e q u a t i o n 2 to give Ki = 10.2 + 1.3 n M . o, E x p e r i m e n t a l d a t a , _ _ _ t h e o r e t i c a l fine. R i g h t p a n e l : p r o g r e s s c u r v e s f o r r a t t y p e 2 50dR w e r e fit i n d i v i d u a l l y to e q u a t i o n 3 a n d a n a l y s e d a s d e s c r i b e d in M a t e r i a l s a n d m e t h o d s to give t h e s e c o n d o r d e r r a t e c o n s t a n t kflKi = 1.37 _+ 0.31 × 106/~[]s. o, E x p e r i m e n t a l d a t a ; _ _ _ , t h e o r e t i c a l fine.
Inhibition of the Rat 5ct-Reductases by Finasteride good agreement with results presented previously with rat prostate 5~R [6, 36]. Progress curves conducted with rat type 2 5~tR yielded different results. As indicated in Fig. 4, inhibition by finasteride appeared to be time dependent. A fit of these time courses to scheme 1 indicates that finasteride forms a preliminary Michaelis complex with the enzyme and N A D P H with Ki = 1.19_+ 0 . 1 0 n M , which is converted to the high-affinity complex (EI*) with a rate constant k3 = 1.62 + 0.:22 x 10-3/s. T h e second-order rate constant for development of inhibition, which applies at inhibitor concentrations below Ki, is k3/ K i = 1.37 + 0.31 x 10 6 I n / s . T h i s rate constant is comparable to that for the inhibition of the h u m a n type 2 isozyme, measured under somewhat different conditions [29].
61
A
3H
I 0
I 5
I 10
I 15
I 20
I 25
Retention Time (rain)
Finasteride
B
Evidence of N A D P + adduct formation by rat type 2 5~R Results from these laboratories indicate that finasteride is a mechanism-.based inhibitor of the h u m a n 5~Rs [29]. Accepted as an alternative substrate, it is ultimately reduced to dihydrofinasteride, but this takes place through an e n z y m e - b o u n d N A D P - d i h y drofinasteride adduct. T h e adduct is released so slowly from the enzyme that the inhibition is essentially irreversible. T h e m e c h a n i s m of inhibition and structure of this adduct are shown in Fig. 1. Studies were conducted with rat type 2 5~R and [3H]-finasteride in order to address whether the same m e c h a n i s m could explain the time-dependent inhibition of this i s o z y m e It was found that inhibition with [3H]-finasteride resulted in an enzyme-inhibitor complex that was not reversible by dialysis. H e a t denaturation of this complex produced two [3H]-products which were readily separated by H P L C [Fig. 5(A)]. T h e polar material eluting in the void volume of the column m a y represent the N A D P [3H]-dihydrofinasteride adduct previously identified with the h u m a n 5~Rs. Additional studies are necessary to confirm this assignment. T h e [3H]-product eluting at 14 min migxates with the same retention time of dihydrofinasteride. Co-injection with authentic [3H]-finasteride (retention time 12.8 min) definitively established th,'tt neither product co-migrated with untransformed inhibitor [Fig. 5(B)]. U p o n heat denaturation, the percentage conversion to dihydrofinasteride (67% in 3 0 m i n ) is consistent with that observed previously wiEh the h u m a n isozymes [29]. In contrast to these results with the rat type 2 isozyme, this m e c h a n i s m does not appear to contribute significantly to the inhibition of rat type 1 isozyme by finasteride. T h e r e is no evidence of time-dependent inhibition (Fig. 4), which argues very strongly that finasteride is a simple, competitive, rapidly reversible inhibitor of this isozyme. Moreover, there is no detectable production of [3H]-dihydrofinasteride in solutions of the enzyme, N A D P H and [3H]-finasteride (data not shown).
I
I
I
I
I
I
0
5
10
15
20
25
Retention Time (rain)
Fig. 5. C h r o m a t o g r a p h i c analysis o f 3H r e l e a s e d f r o m rat type 2 50d1 after i n a c t i v a t i o n by [3H]-finasteride. (A) E n z y m e : i n h i b l t o r c o m p l e x w a s p r e p a r e d b y i n c u b a t i n g rat type 2 5~R with [3H]-finasteride. T h e s a m p l e w a s dialysed a n d heat d e n a t u r e d for 30 m i n . A portion o f the s a m p l e w a s a n a l y s e d b y r e v e r s e - p h a s e H P L C . T h e two p r o d u c t s elute with retention t i m e s o f ~4 m i n a n d 14 rain. (B) Co-injection d e m o n s t r a t e s that a u t h e n t i c [3H]-finasteride (retention t i m e , 1 2 . 8 m i n ) does not c o - m i g r a t e with the p r o d u c t s r e l e a s e d f r o m the enzyme.
DISCUSSION
Finasteride was originally described as a reversible, competitive inhibitor with a Ki value ~ 6 n M for the 5~R in rat prostate and liver [6, 36]. At that time it was thought that there was a single form of 5c~R [4]. N o r m i n g t o n and Russell [20] definitively established that there are two genes for 5~R in the rat encoding what are now defined as type 1 and 2 5c~R. T h e presence of two isozymes in the rat warranted a re-examination of the m e c h a n i s m of inhibition of the individual isozymes by finasteride because it is unclear which isozyme was previously studied. Initial work in our laboratory was conducted with native sources (i.e. tissue extracts) of the rat 5~Rs. T h e difference in p H dependence of the isozymes was exploited to enhance the sensitivity for rat types 1 and 2 5c~R [20]. F r o m these studies it was concluded there were differences in the m e c h a n i s m of inhibition of rat types 1 and 2 5aR by finasteride; however, definitive studies required the use of recombinant 5~Rs to ensure that
62
B. Azzolina
the enzyme preparation was homogeneous with respect to isozyme content. As reported in this paper, studies using both intact cells and enzyme preparations of the isozymes confirmed that finasteride is a reversible inhibitor of rat type 1 and a time-dependent inhibitor of rat type 2 5~tR. T h e finding that finasteride is a reversible inhibitor of rat type 1 isozyme is in good agreement with the original reports concerning the m e c h a n i s m of inhibition of 5ctR by this c o m p o u n d . It is reasonable to assume that under the neutral p H conditions of those studies type 1 5~R activity predominated; therefore conclusions drawn from early studies are consistent with those reported in this p a p e r that finasteride is a reversible, competitive inhibitor. Furthermore, it is now understandable why the more complicated m e c h anism-based inhibition for the h u m a n 5~Rs did not come to light until recombinant enzymes were available. Most early kinetic studies were conducted with 5ctR from the rat liver because this tissue was readily available and contained >100-fold higher levels of enzyme activity c o m p a r e d to h u m a n tissues [8, 37]. Rat liver is enriched in rat type 1 5~R and accordingly reversible inhibition by finasteride was observed. T h e r e is good evidence that finasteride displays time-dependent inhibition of rat type 2 5~R (Figs 2 and 4). However, this observation alone provides little insight into the details of the m e c h a n i s m of inhibition. Previous studies with the h u m a n 5c~Rs relied on product identification after incubation with [3H]-finasteride to dissect the m e c h a n i s m of inhibition shown in Fig. 1 [29]. T h e chromatographic and spectroscopic properties of the material released from the h u m a n enzymes were consistent with an N A D P adduct. Furthermore, heat denaturation of the h u m a n 5~R-inhibitor complex liberated dihydrofinasteride, the structure of which was confirmed by mass spectroscopy. A similar approach was used to assess the inhibition of the rat type 2 5~R by finasteride. H e a t denaturation of the rat enzyme-[3H]-inhibitor complex produced two products which migrated by reverse-phase H P L C as expected for the N A D P adduct and dihydrofinasteride (Fig. 5). Additional characterization was not possible because of the limited amounts of rat type 2 5~R produced by transient transfection in C O S cells. However, these preliminary findings with rat type 2 5~R, in the light of the results with the baculovirus-expressed h u m a n 57Rs, suggest that mechanism-based inhibition is involved in the inactivation of rat type 2 isozyme by finasteride. T h e reversible inhibition of rat type 1 5~R by finasteride is somewhat surprising given that m e c h a n i s m based inhibition by this c o m p o u n d has now been established for rat type 2, h u m a n type 1 and h u m a n type 2 5erRs [29]. Why is finasteride unable to inactivate rat type 1 5~R irreversibly? T h e first step in the m e c h a n i s m presented in Fig. 1 requires that enzymes recognize finasteride as an alternative substrate and
et al.
catalyse reduction of the double b o n d in the A-ring of the azasteroid. I f the enzyme is incapable of this reaction, reversible inhibition by finasteride will result. Alternatively, reduction of finasteride could occur, but the partitioning of the enolate might favor proton transfer to yield dihydrofinasteride rather than collapse with N A D P ÷ to form the stable adduct (see Fig. 1). In order to distinguish between these possibilities, rat type 1 5ctR was incubated with 3H-finasteride and the product(s) analysed by H P L C . N o conversion of finasteride to dihydrofinasteride was detected. Therefore, finasteride is a reversible inhibitor of rat type 1 5~R because the enzyme is unable to catalyse the reduction of finasteride, the first step required for mechanism-based inhibition. It is worth noting that rat and h u m a n type 1 5~Rs appear to be more closely related (60% sequence identity) than are the h u m a n type 1 and type 2 isozymes (50%). However, despite the lower sequence homology of the h u m a n isozymes and the p o o r inhibition of h u m a n type 1 5c~R by finasteride (Table 1), mechanism-based inhibition of b o t h h u m a n homologs is observed. T h e distinct m e c h a n i s m of inhibition found with rat type 1 5c~R suggests that a subtle difference exists in the N A D P H - b i n d i n g site of this isozyme which interferes with enzyme-catalysed reduction of A1-4-azasteroids but not A4-3-keto-steroids (e.g. T). As noted previously, 5~R does not contain a consensus N A D P H binding site [38]. Furthermore, this class of enzymes has been intractable to purification and consequently no structural information is available. However, structure-function information was inferred from characterization of mutations in the h u m a n type 2 gene from subjects with the inherited 5ctR defciency [38]. T w o amino acids were proposed to contribute to the T binding site; both of which are conserved a m o n g isozymes and species. Eight amino acids were tentatively identified as contributing to the N A D P H - b i n d i n g site. Strict conservation is observed in seven of these residues when protein sequences of types 1 and 2 5~R from rat, cynomolgus and h u m a n are c o m p a r e d [19-21, 39]. Interestingly, arginine at position 145 of the h u m a n type 2 protein is conserved in all isozymes with the exception of rat type 1 where cysteine is found at the corresponding residue. Whether substitution at this position contributes to the different m e c h a n i s m of inhibition by finasteride remains to be determined. CONCLUSION Finasteride is a potent inhibitor of both types 1 and 2 5~R in rat. As described in this paper, species differences in the inhibition by finasteride are seen in both potency and in the m e c h a n i s m of the inhibition. Finasteride is a mechanism-based inhibitor of the h u m a n types 1 and 2 5c~R with >100-fold selectivity for h u m a n type 2 [29]. Surprisingly, finasteride is a
Inhibition of the Rat 5ct-Reductases by Finasteride
potent, reversible inhibitor of the rat type 1 57R (IC50 13 nM) but a m e c h a n i s m - b a s e d inhibitor of the rat type 2 isozyme (ICso 1 nM). In the light of these findings, one needs to consi~der whether the biological activity of finasteride in tl~e various in vivo rat models is caused by the inhibition of b o t h isozymes of 5~R.
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