Biochem. J. (1986) 240, 717-723 (Printed in Great Britain)

Active-site directed inactivation of rat ovarian 20a-hydroxysteroiddehydrogenaseJoseph W. RICIGLIANO and Trevor M. PENNING*Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, U.S.A.

Rat ovarian 20a-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition bycatalysing the reduction of progesterone to give the progestationally inactive steroid 20a-hydroxypro-gesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestationalmaintaining agents, including the epimeric allylic alcohol pair 3/J-hydroxy-a-vinyl-5a-androstane-177fl-methanol and the related vinyl ketone 1-(3fl-hydroxy-5a-androstan-17,8-yl)-2-propen-1-one. The vinylketone inactivates rat ovarian 20a-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarosecolumn chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivationplots gave a Ki of 2.0 gSM for the inhibitor and a ti for the enzyme of 20 s at saturation. These data indicatethat the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis inthe presence or absence of a competing nucleophile nor gel filtration reverses the inactivation, suggestingthat a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20a-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocitymeasurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketonecan behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivationexperiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrastthe allylic alcohol pair 3f-hydroxy-a-vinyl-5a-androstane-17,8-methanol are neither substrates nor inhib-itors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivationobserved with the vinyl ketone suggests that this compound may be useful as a progestational maintainingagent.

INTRODUCTION

20a-Hydroxysteroid dehydrogenase plays a centralrole in progesterone metabolism, catalysing the NADP+linked reduction of progesterone (Wiest, 1959; Wiest &Wilcox, 1961) to yield the progestationally inactivemetabolite 20a-hydroxyprogesterone (Talwalker et al.,1966). The ovarian enzyme plays a major role in thetermination of pregnancy in the rat (Wiest, 1968), wherea 50-fold increase in activity is observed at parturition(Kuhn & Briley, 1970). Similarly a 1.5-fold post-partumincrease has been noted in human placental 20a-hydroxysteroid dehydrogenase (Diaz-Goya et al., 1979).These increases in 20a-hydroxysteroid dehydrogenaseactivity rather than decreased progesterone synthesiscontribute to the lower plasma progesterone levelsassociated with the termination of pregnancy. Theovarian enzyme also catalyses the decline in progesteronelevels which occurs during leuteolysis (Lamprecht et al.,1969). Because of its apparent role as a regulator ofprogesterone levels in the cycling and pregnant female,20a-hydroxysteroid dehydrogenase represents a targetenzyme for drug action. Potent selective inhibitors of thedehydrogenase have the potential to maintain progester-one levels and perhaps prevent miscarriage.

In considering candidates that might promote irrever-sible inhibition of the ovarian 20a-hydroxysteroiddehydrogenase we were attracted to the previous studies

of Maclnnes on alcohol dehydrogenase (Maclnnes et al.,1981). These studies showed that alcohol dehydrogenasecatalyses the oxidation of allylic alcohols to yield thecorresponding Michael acceptors (a,fl-unsaturated alde-hydes), which, when generated within the proximity oftheactive site, can undergo alkylation to produce inactivatedenzyme. Employing a similar strategy Covey (1979)synthesized an epimeric pair of 1 7,8-propargylic alcohols:17fl-[(1 S)-1-hydroxy-2-propynyl]androst-4-en-3-one and17,1-[(1R)-l-hydroxy-2-propynyl]androst-4-en-3-one aspotential suicide substrates of20a-hydroxysteroid dehyd-rogenase. It was subsequently found that the I Sstereoisomer acts as a suicide substrate of 20,1-hydroxysteroid dehydrogenase form Streptomyces hy-drogenans (Strickler et al., 1980), whereas the IRstereoisomer acts as a suicide substrate of the 17,/20a-hydroxysteroid dehydrogenase of human termplacenta (Tobias et al., 1982). The ability of thesecompounds to inactivate ovarian 20a-hydroxysteroiddehydrogenase has never been reported. One drawbackto these studies is that the propargylic alcohols containconjugated ketones in their A-rings and may displayaffinity for the progesterone receptor. By contraststeroids in the Sa- and 5,8-pregnane series have noprogestational activity (Ogle & Beyer, 1982) and havebeen reported to display a high affinity for rat ovarian20a-hydroxysteroid dehydrogenase (Wilcox & Wiest,1966). We have exploited these earlier findings and

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Abbreviations used: 20a-hydroxyprogesterone, 20a-hydroxypregn-4-en-3-one; vinyl ketone, 1-(3,8-hydroxy-5ca-androstan- 17,8-yl)-2-propen- I -one;allylic alcohol, 3fl-hydroxy-a-vinyl-5a-androstane-17,8-methanol.

* To whom all correspondence and reprint requests should be sent.

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J. W. Ricigliano and T. M. Penning

CH3 0 1. Pb(OAc)4 H 02. NaBH43. 104-

HO HHO H

Al lopregnanolone

H0o H

5. JonesH reagent

O >~i~ HO 2I

(I) 4. CH2=CHMgBr

f HH

HO HHO

(111) (11)Scheme 1. Synthesis of the racemic allylic alcohol pair (II) and

the vinyl ketone (III)

describe here the synthesis and evaluation of threeSa-steroids which contain either a vinyl ketone or anallylic alcohol in place of the side chain at C-17. Onesuch compound, 1 -(3,l-hydroxy-Sa-androstan- 17,/-yl)-2-propen-l-one (Scheme 1: III), is an exceedingly potentand efficient inhibitor of the rat ovarian 20a-hydroxy-steroid dehydrogenase; its potential as a progestationalmaintaining agent is discussed.

steroid was increased by a factor of 3. N.m.r. indicatedthat aldehyde I had been formed via this route, since analdehyde proton with a chemical shift of 9.66 p.p.m. wasobserved. Other chemical shifts agree with those reportedby Gelbart & Thomas (1978).

Synthesis of 3/I-hydroxy-a-vinyl-5a-androstane-17/I-methanol (II) and1-(3p8-hydroxy-5a-androstan-17fi-yl)-2-propen-1-one(III)Aldehyde I (200 mg, 6.6 mmol) and 15 ml of dry

dioxane (CaH2 distilled) were placed in an oven-dried,round-bottomed flask fitted with a septum and thesystem was purged with dry N2. Vinyl magnesiumbromide (4 ml of 1.0 M solution in tetrahydrofuran) wasintroduced via a syringe. Once hydrogen evolution hadceased (3 min) the reaction was placed on ice andquenched with 2 ml of water and 0.1 ml of2.0 M-sulphuric acid. After the bulk of solvent had beenremoved under reduced pressure, the reaction mixturewas poured into water and extracted with chloroform.Extracts were washed with water and concentrated todryness. The total products were dissolved in methanol,applied to semi-preparative t.l.c. plates and developed intoluene/acetonitrile (10:3, v/v). Product II, whichconsisted of both epimeric alcohols, was eluted withmethanol. Ir.: (cm-') -OH, 3610 m, 3400 s and b,1028 s; -CH=CH2, 991 s, 953 m, 926 m and b; steroidnucleus, 2930 s, 2861 s, 1448 s, 1380 s. N.m.r.: & (p.p.m.)([2H]chloroform) 0.66 and 0.77 (2 s, C-18 -CH, peakheight ratios 4:5), 0.80 and 0.82 (2 s, C-19 -CH,,, peak

MATERIALS AND METHODS

3,8-Hydroxy-5a-pregnan-20-one (allopregnanolone)was purchased from Steraloids Inc. (Wilton, NH,U.S.A.). NADP+ and 20a-hydroxyprogesterone wereobtained from Sigma Chemical Co. (St. Louis, MO,U.S.A.). Poly(L-lysine)-agarose was purchased fromP and L Biochemicals (Milwaukee, WI, U.S.A.) andSephadex G-25F was obtained from Pharmacia(Piscataway, NJ, U.S.A.). All solvents were purchasedfrom Burdick and Jackson (Muskegon, MI, U.S.A.), ex-cept toluene, which was obtained from Fisher Scientific.Other reagents were products of Aldrich Chemical Co.(Milwaukee, WI, U.S.A.).

All n.m.r. were recorded on a Brucker WM-360 MHzspectrometer, in C2HCl3 with a deuterium field lockrelative to tetramethylsilane. Infrared spectra wererecorded on a Perkin-Elmer 521 spectrometer with anextended range interchange. Absorption bands are listedas strong (s), medium (m), weak (w) and broad (b).Melting points were taken on a Fisher-Johns melting-point apparatus and were not corrected. Reactions werefollowed by t.l.c. carried out on Analtech 02521 plates(Analtech Inc., Newark, DE, U.S.A.). Semi-preparativet.l.c. was performed on 20 cm x 20 cm E. Merck 5763plates (Darmstadt, E. Germany) in the solventsindicated.

Steroid synthesisPreparation of the intermediate aldehyde 3,/-hydroxy-

17,f-formyl-androstane (I) (Scheme 1) was achieved byusing a modification of the method described by Gelbart& Thomas (1978). In order to increase the yield of thecrucial acetoxylation product (3,6,21-diacetoxy-5a-pregnane-20-one) to 80%, the ratio of Pb(OAc)4 to

co"; 1.1

80 _E0C

60 'E

0EC

40 -U~0m

20 aaz

Fraction no.Fig. 1. Purification of rat ovarian 20a-hydroxysteroid dehydro-

genase by poly(L-lysine)-agarose chromatography

Rat ovarian cytosol (4 ml obtained from 10 ovaries) wasapplied to a 2 ml column of poly(L-lysine)-agaroseequilibrated in 10 mM-potassium phosphate containing12 mM-monothioglycerol and 1 mM-EDTA, pH 6.0. Thecolumn was washed in equilibration buffer followed by100 mM-NaCl dissolved in the equilibration buffer asindicated. The absorbance at 280 nm ( ) and theenzyme activity (0 O) were monitored, and thefractions indicated were pooled.

1986

718

Inactivation of 20a-hydroxysteroid dehydrogenase

-00

.)

(Ua)ENc0)

70

60~

50

-4

20

10

5 10 15 20 25 30 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7Time (min) 1/[Vinyl ketone] (pM)-1

Fig. 2. Time-dependent inactivation of rat ovarian 20a-hydroxysteroid dehydrogenase mediated by vinyl ketone (III)

Portions (192 u1; 0.52 mg of protein/ml) of semi-purified thiol-free rat ovarian 20a-hydroxysteroid dehydrogenase wereincubated in a final volume of200 ,1 containing the concentration ofvinyl ketone indicated in4% acetonitrile. Aliquots (10-20 ,ul)were removed at various times and diluted directly into the standard 1.0 ml assay for measurement of enzyme activity (a). Thetimes required to promote 50% loss ofenzyme activity (t1) were determined from estimates of the initial velocities of inactivation;these values were then plotted against the concentration of vinyl ketone (b). The line shown was drawn by regression; r = 0.94.

height ratio 4: 5), 3.6 (m, CHOH, C-3); chemical shifts forthe side chains are: 5.84 (1H, m, -CHC), 5.06 (1H,q, C=CH2), 5.02 (1H, q, C=CH2), 3.98 (1H, m, -CH-CH=).

Oxidation of the allylic alcohols (II) (100 mg) to thevinyl ketone (III) was performed in acetone with excessJones reagent (CrO3/H2SO4/water). The reaction wasquenched with methanol followed by dilution with water.The product was extracted in chloroform, concentratedto dryness and applied to semi-preparative t.l.c. plates ina methanol solution. Plates were developed in toluene/acetonitrile (10:2, v/v). The vinyl ketone product (III)was visualized under u.v. light, eluted with methanol andcrystallized from hexane/acetone. I.r.: (cm-') C0O,1690 s and b (shoulder at 1658), 1102 s; C=CH2,1603 s, 983 s, 965 m; steroid nucleus, 2925 s, 2852 s,1438 s, 1369 m. U.v. maximum 216 nm in methanol, 62167000. N.m.r.: a (p.p.m.) ([2H]chloroform) 0.61 (3H, s,C-18 -CH3), 1.03 (3H, s, C-19 -CH3), 6.44 (1H, q,J2,3cis 10.8 Hz, trans 16.6 Hz, -CO-CHC), 6.22 (1H, d,

J2 3 trans 16.6 Hz, CH=CH2), 6.44 (1H, d, J2 3 cis10.8 Hz, CH=CH2). M.p. 165-167 'C.

Enzyme studiesPreparation of 20a-hydroxysteroid dehydrogenase. In

a typical preparation five female Sprague-Dawley rats(150-200 g; Charles River Breeding Laboratories, Wilm-ington, MA, U.S.A.) were killed by cervical dislocationat random phases of the oestrus cycle. Their ovaries wereexcised, washed in ice-cold 0.25 M-sucrose and homo-genized in 4.0 ml of 10 mM-potassium phosphate contain-ing 12 mM-monothioglycerol and 1 mM-EDTA, pH 6.0.Cytosol was then prepared by differential centrifugationand chromatographed on a 2 ml column of poly(L-lysine)-agarose equilibrated in homogenization buffer.After the column was washed free of non-absorbedprotein the dehydrogenase was eluted with homogeniza-tion buffer containing 100 mM-NaCl. The active fractionswere pooled and dialysed against three changes (1: 100,

v/v) of 10 mM-Tris/HCl containing 12 mM-monothio-glycerol, 1 mM-EDTA and 20% (v/v) glycerol, pH 8.0.Aliquots (1.0 ml) containing 0.6 mg of protein/ml werefrozen in liquid N2 and stored at -80 °C for future use.Several separate preparations ofenzyme were used in thisstudy and their specific activities ranged from 105 to180 nmol of 20a-hydroxyprogesterone oxidized/minper mg of protein. For details of the enzyme assay seebelow.

Assay of 20a-hydroxysteroid dehydrogenase activity.Enzyme activity was measured in 1.0 ml systemscontaining 0.1 ml of 1.0 M-Tris/HCl, pH 8.0, 20 u1 of115 mM-NADP+, 20 ,u of 2.2 mM-20a-hydroxypro-gesterone, 20 1l of acetonitrile and water. The finalconcentration oforganic solvent was 4% (v/v). Reactionswere initiated by the addition ofenzyme and followed bymeasuring the production of reduced pyridine nucleotideat 340 nm (6340 6270 M-1 cm-') on a Beckman DU-7spectrophotometer at 25 'C. Activities were corrected forthe non-enzymatic reduction of nucleotide; this rate wastypically no more than 2-5% of the enzyme-catalysedreaction.

Inactivation of 20a-hydroxysteroid dehydrogenase.Samples of frozen enzyme (1.0 ml, 0.6 mg of protein/ml)were thawed and dialysed against three changes (1: 1000,v/v) of 10 mM-Tris/HCl, pH 8.0, to remove the mono-thioglycerol. After dialysis the thiol-free enzyme retained80% of its original activity. Portions of the dialysedenzyme (192,l) were placed in a final volume of 200,tlcontaining various concentrations of steroid inactivator(0-33.3 ftM) in 4% acetonitrile at 25 'C. At time intervalsthereafter portions (10-20 gtl) were withdrawn from theincubation and diluted directly into the 1.0 ml assaysystem. The high concentration of 20a-hydroxypro-gesterone (44 ,tM) and NADP+ (2.3 mM) in the assay andthe extensive dilution (50-100-fold) effectively quenchany further alkylation events. The determination of

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(b)

(b)

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J. W. Ricigliano and T. M. Penning

Table 1. Dialysis of rat ovarian 20a-hydroxysteroid dehydro-genase inactivated by the vinyl ketone (111)

Two identical (500,1, 0.77 mg of protein/ml) samples ofthiol-free rat ovarian 20a-hydroxysteroid dehydrogenasewere treated with either 4% acetonitrile (Control) or40 /LM-vinyl ketone in 4% acetonitrile (Experimental).Aliquots of enzyme were removed after 20 min andassayed for enzyme activity. Each sample was then dividedinto two portions and dialysed extensively against either10 mM-Tris/HCl, pH 8.0, or 10 mM-Tris/HCl containing0.25 mM-2-mercaptoethanol, pH 8.0. Aliquots were with-drawn from the dialysis after 5 and 14 h and assayed forenzyme activity. All measurements have S.D. of9% or less.

Enzyme activity (%)

Experi-Manipulation Control mental

No addition 100 1004% acetonitrile after 20 min 9440 ,uM-vinyl ketone after 20 min 7Dialysis5h 88 414h 82 35 h (plus 2-mercaptoethanol) 97 614 h (plus 2-mercaptoethanol) 90 10

0.20 (a)

0.15

0.10

0.05

(b)

0.15-

0.10

0.05-

011 3 5 7 9 11 13

Fig. 3. Gel exclusionhydroxysteroidketone (III)

30

20

10 I

0 .tU

a)30 E

NC

20 .4'

10

0

Fraction no.chromatography of rat ovarian 20a-dehydrogenase inactivated by the vinyl

enzyme activity in these assays is thus a measure ofenzyme inactivation. Semi-logarithmic plots of initialenzyme activity (per cent) versus time gave estimates ofti for inactivation. Secondary plots of ti versus 1 /[I] gavea tq for the enzyme at saturating inhibitor concentrationand a Ki for the inhibitor. Control incubations, whichcontained acetonitrile only, indicated that the enzymeretains full activity during the time course of enzymeinactivation.

Protein determinationProtein concentrations were determined by the method

of Bradford (1976), employing crystalline bovine serumalbumin (Armour Pharmaceuticals, Kankakee, IL,U.S.A.) as a standard.

Portions (0.55 ml, 0.45 mg of protein/ml) of semi-purifiedthiol-free rat ovarian 20a-hydroxysteroid dehydrogenasewere incubated in the presence of 40 /M-vinyl ketonedissolved in 4% acetonitrile or 4% acetonitrile alone for20 min at 25 'C. At this time less than 10% of the initialenzyme activity remained in the sample containing vinylketone. The incubation mixtures were then applied to acolumn of Sephadex G-25F (0.8 cm x 46 cm) equilibratedin 1O mM/Tris-HCl containing 12 mM-monothioglyceroland 1 mM-EDTA, pH 8.0. The absorbance at 280 nm wasmonitored ( ) and enzyme activity in each fractiondetermined (@---M). The elution profiles observed withenzyme incubated in the absence of vinyl ketone (a) andthe presence of vinyl ketone (b) are shown. In both casesthe enzyme elutes in the void volume, which correspondsto fraction no. 7.

by Pongswadii & Anderson (1984), and a Vmax. valueRESULTS identical with our specific activity measurement.

Purification and properties of 20a-hydroxysteroiddehydrogenaseWhen rat ovarian cytosol is subjected to poly(L-lysine)-

agarose chromatography the 20a-hydroxysteroid dehy-drogenase is separated from the majority of cytosolicprotein (Fig. 1). The result is similar to that reported byMori & Wiest (1979). The pooled active fractionsroutinely gave a specific activity of 120 nmol of20a-hydroxyprogesterone oxidized/min per mg, whichrepresents a 5-7-fold protein purification. Specificactivities reported for the purified enzyme range from1990 to 8317 nmol of 20a-hydroxyprogesterone oxid-ized/min per mg (Pongsawasdi & Anderson, 1984; Mori&Wiest, 1979). Based on these measurements our prepara-tion is 1-5% pure. Determination ofkinetic constants forour preparation gave a Km of2.0 UM for 20a-hydroxypro-gesterone, which is in agreement with the value reported

Inactivation of 20a-hydroxysteroid dehydrogenaseUpon incubation of the semi-purified 20a-hydroxy-

steroid dehydrogenase with various concentrations of thevinyl ketone (III), rapid time-dependent loss of enzymeactivity is observed (Fig. 2a). This inactivation isconcentration-dependent and demonstrates saturationkinetics. To obtain reliable kinetic constants for theinactivation event, a large number of time points weretaken in the first minutes of each time course (completedata not shown). When these results are transformed byplotting the t1 (s) against the reciprocal of the inhibitorconcentration the resulting plot indicates that the vinylketone is a potent inhibitor (Ki = 2.0 /SM) and a veryrapid inactivator, yielding a ti for the enzyme of 20 s atsaturation (Fig. 2b). This method of data analysis isessentially identical with that employed by Kitz & Wilson(1962).

1986

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0C4c

;lq

Inactivation of 20a-hydroxysteroid dehydrogenase

0 2 4 6 8 10 12 14

Time (min)

.-1

C._4 )

ENc

4-

0 2 4 6 8 10 12 14

Time (min)

Fig. 4. 20a-Hydroxyprogesterone and NADP+ protect rat ovarian 20a-hydroxysteroid dehydrogenase from inactivation mediated bythe vinyl ketone (III)

Portions (20 ,d, 0.47 mg of protein/ml) of semi-purified thiol-free rat ovarian 20a-hydroxysteroid dehydrogenase were incubatedin the presence of 5.0 /LM-vinyl ketone and increasing amounts of 20a-hydroxyprogesterone (0-25 /sM) as indicated. Enzymeactivity in the absence ofany steroid is also shown. The concentration oforganic solvent was maintained at 4% (v/v) acetonitrile.Aliquots (20 ,ul) were removed with time and diluted directly into the standard 1.0 ml assay for determination ofenzyme activity(a). Portions (20 ll, 0.44 mg of protein/ml) of semi-purified thiol-free rat ovarian 20a-hydroxysteroid dehydrogenase were

incubated in the presence of 8.6 #M-vinyl ketone and increasing amounts of NADP+ (0-200 ,uM) as indicated. Enzyme activityin the absence of any additions is also shown. Aliquots (20,1) were removed at the indicated time and diluted directly intothe standard 1.0 ml assay for determination of enzyme activity (b).

Evidence that the vinyl ketone (Ill) forms a stablecovalent bond with 20a-hydroxysteroid dehydrogenaseThe time-dependent loss of enzyme activity mediated

by the vinyl ketone (III) shown in Fig. 2(a) was followedafter substantial dilution of the reaction mixture(50-100-fold). To establish that this inactivation eventresulted in covalent bond formation, preparations of thedehydrogenase inactivated by this compound weresubjected to exhaustive dialysis (Table 1). Theseexperiments indicate that enzyme activity does not returnafter this treatment. Moreover, dialysis in the presence ofa powerful competing nucleophile (2-mercaptoethanol)was unable to displace the steroid from the protein andrestore enzyme activity. During these manipulationscontrol enzyme retained at least 80% of its initialactivity.

Additional evidence for covalent bond formation wasobtained by subjecting samples of control enzyme andenzyme inactivated with vinyl ketone (III) to gel-exclusion chromatography (Fig. 3). In both instances theenzyme elutes in the void volume but activity is notrestored to the inactivated preparation.

The vinyl ketone (III) is an active-site directedalkylating agentBoth the substrates, 20a-hydroxyprogesterone and

pyridine nucleotide (NADP+), provide protection frominactivation mediated by the vinyl ketone (III) (Fig. 4a

and 4b). Secondary plots of l/kapp. versus the concen-tration of protecting ligand provide estimates of thedissociation constants of 20a-hydroxyprogesterone(Kd = 1.6 /LM) and NADP+ (Kd = 1.6 /tM). Although wehave not independently obtained a Kd for the steroidalsubstrate, the Kd for NADP+ is in close agreement withthat obtained by Pongsawasdi & Anderson (1984). Tosubstantiate the idea that the vinyl ketone competes forthe active site of the enzyme we have also shown that thecompetitive inhibitor of the dehydrogenase, 17,?-oestradiol, will protect the enzyme from inactivation. Inthese protection studies a Kd of 5.0 /tM was obtained for17,-oestradiol, which is identical with that obtained forthis compound in our competitive inhibition studies(data not shown). Furthermore, under initial velocityconditions in which the concentrations of 20a-hydroxy-progesterone (44 /tM) and pyridine nucleotide (2.3 mM)are able to provide complete protection against inactiv-ation, the vinyl ketone is able to act as a competitiveinhibitor of the dehydrogenase (Fig. 5). Under suchconditions the vinyl ketone is presumably binding to theenzyme in a rapid reversible fashion, but it does notoccupy the active site long enough to initiate analkylation event. In these competitive inhibition studiesthe Ki value determined for the vinyl ketone is 2.0 /tM andis identical with the Ki determined in the inactivationexperiments. Together these data provide a strong casefor the view that the vinyl ketone is an active-sitedirected agent.

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0-0

4-1

C._

ENc

a)

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J. W. Ricigliano and T. M. Penning

0 10 20 30 40 50

[Vinyl ketone] (pM)Fig. 5. Competitive inhibition of the initial rate of 20a-

hydroxyprogesterone oxidation by the vinyl ketone (Il)Initial velocities of NADP+ reduction at fixed concentra-tions of 20a-hydroxyprogesterone (5-40,M) were deter-mined in the presence of various concentrations of vinylketone (0-40 /SM); in each instance the reaction wasinitiated with 11 ,ug of enzyme. The data were analysed bya primary Dixon plot (Dixon, 1953). The inset shows thesecondary Dixon plot (Segal, 1975) from which the Ki wasdetermined. Abscissa values of the secondary plot are theslopes determined for the lines of the primary plotrepresented as (nmol of NADP+/min per mg ofprotein)-1/uM-vinyl ketone; ordinate values refer to theconcentration of 20ca-hydroxyprogesterone (,m-1).

Evaluation of the C-20 epimeric alcohols143/i-hydroxy-5a-androstan-17fl-yl)prop-2-en-l-one (U)as inhibitors of 20a-hydroxysteroid dehydrogenaseThe C-20 epimeric alcohols (II) are synthetic inter-

mediates on the pathway to the vinyl ketone (III). It wasfelt that the C-20 a-allylic alcohol could be oxidized by thedehydrogenase to the corresponding vinyl ketone, which

could then act as a Michael acceptor and inactivate theenzyme. In this manner the allylic alcohols would act assuicide substrates in a fashion similar to that describedby Maclnnes et al. (1981) for alcohol dehydrogenase.Evaluation of the racemic mixture of C-20 epimericalcohols indicated that they cannot be utilized assubstrates even when highly favourable conditions foroxidation were employed (i.e. pH 10.0 and 0.4 mg ofenzyme protein to initiate the reaction). Moreover, theracemic mixture was unable to inhibit the enzyme-catalysed oxidation of 20a-hydroxyprogesterone even atthe limit of steroid solubility (approximately 50 ,M),suggesting that these compounds are excluded from theactive site at these concentrations. Similarly the vinylketone (III) was examined as a substrate by optimizingconditions for reduction (i.e. pH 6.0 and 0.4 mg ofprotein to initiate the reaction): under these conditionsno consumption of NADPH could be measured.

DISCUSSIONDecreases in plasma progesterone levels generally

precede the termination of pregnancy. This is truewhether progesterone at term is produced by the corpusluteum as in the rat (Caspo & Wiest, 1969) or in theplacenta as in sheep (Bassett et al., 1969) and humans(Caspo, 1977). Evidence exists that 20a-hydroxysteroiddehydrogenase is a major enzyme responsible for thedecline in progesterone levels since it catalyses thereduction of this hormone to give the inactive 20a-hydroxyprogesterone. A rationale thus exists for thedevelopment of selective inhibitors of the 20a-hydroxy-steroid dehydrogenase as potential progestationalagents. This paper indicates that the vinyl ketone (III) isa potent (Ki = 2.0,M) and exceedingly efficient inacti-vator of the dehydrogenase, yielding a ti for the enzymeof 20 s at saturation. Moreover, the compound isactive-site directed and forms a stable covalent bond withthe enzyme. The mechanism of inactivation is believed tooccur via nucleophilic attack of the vinyl ketone (Scheme2). This represents another example of how theintroduction of Michael acceptors placed on the steroidnucleus can yield highly reactive alkylating agents(Penning et al., 1981). Further it can be speculated thatthe reason for the rapid inactivation may be polarizationof the C-20 carbonyl, which may be assisted by apositively charged amino acid at the active site. Such amechanism would facilitate hydride transfer in thenormal catalytic cycle of the enzyme.

It will be recalled that Covey (1979) has synthesizeda progesterone analogue (17,-[(lR)-l-hydroxy-2-pro-pynyl]androst-4-en-3-one) containing a propargylic al-

H

0

ENZ-Y H+

@ o2_H IENZ

H oH

0 0

Scheme 2. Proposed mechanism of inactivation of the ovarian 20a-hydroxysteroid dehydrogenase by the vinyl ketone

1986

H H

OH

4y/ ENZ

go,.J,H

722

Inactivation of 20a-hydroxysteroid dehydrogenase 723

cohol in its side chain; this compound appears to act asa suicide substrate for the human placental 17/J-/20a-hydroxysteroid dehydrogenase (Tobias et al., 1982).Thus the acetylenic alcohol is oxidized by the enzymeto yield the corresponding acetylenic ketone, which canthen promote a rapid time-dependent inactivation of theenzyme (ti = 1 min at 50/M inhibitor). A more thoroughexamination of this earlier work, however, reveals thatthe acetylenic alcohols are oxidized by the enzyme undernon-physiological conditions (0.5 mM-steroid), suggest-ing that these compounds may act as suicide substratesonly under forced conditions. It should be emphasizedthat the ability of the acetylenic alcohols and ketones toinactivate the ovarian 20a-hydroxysteroid dehydrogen-ase has not been assessed; moreover, the affinity of theseprogesterone analogues (agonist or antagonist) for thehuman progesterone receptor has not been reported.The vinyl ketone (III) described here represents an

exciting development since, unlike the acetylenic alcoholor ketone synthesized by Covey (1979), it is not aprogesterone analogue since it contains a saturatedA-ring ofthe trans configuration. Moreover, it inactivatesthe dehydrogenase with an exceedingly short half-life andit displays a low Ki, suggesting that this compound maybe a superior alkylating agent. The ability of the vinylketone to inhibit the ovarian 20a-hydroxysteroid de-hydrogenase in vivo and act as an anti-leuteolytic agentand maintain pregnancy in the rat remains to be deter-mined. The success of such studies will depend upon theselectivity of the vinyl ketone for the dehydrogenase andhow rapidly this Michael acceptor will be scavenged byendogenous nucleophiles.The ability of the vinyl ketone (III) to inactivate the

human placental 20a-hydroxysteroid dehydrogenasealso remains to be determined. At the present time thereis reason to believe that the 20a-hydroxysteroiddehydrogenase activity of rat ovarian cytosol and humanplacenta may be catalysed by quite different proteins. Inthe placental enzyme both the 17,/- and 20a-hydroxy-steroid dehydrogenase activities co-exist on the sameprotein (Strickler et al., 1981); there is no evidence tosuggest that the same is true for the rat ovarian enzyme.

Unlike the propargylic alcohol and acetylenic ketonesynthesized by Covey (1979) neither the allylic alcoholsnor the vinyl ketone described here appear to besubstrates for the rat ovarian 20a.-hydroxysteroiddehydrogenase. This may not be an unexpected findingsince the Km values reported for the propargylic alcoholswere close to 0.5 mm. At this concentration the allylic

alcohol pair and vinyl ketone described here areinsoluble in the assay system.

This work was supported by BRSG 2-507-RR-05415-21,BRSG 2-507-RR-05415-24 and a Pharmaceutical Manufac-turers Association Research Starter Grant awarded to T. M. P.J. W. R. is a Predoctoral Fellow supported by a CardiovascularTraining Grant Fellowship HL 07502. We thank Dr. KurtLoewing of the Chemical Abstracts Service for steroidnomenclature.

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Received 27 February 1986/7 July 1986; accepted 22 August 1986

Vol. 240