Identification of a First Enzymatic Activator of a 17β-HydroxysteroidDehydrogenaseAlexandre Trottier, Rene Maltais, and Donald Poirier*

Laboratory of Medicinal Chemistry, Endocrinology and Nephrology Unit, CHU de Quebec (CHUL, T4), and Faculty of Medicine,Laval University, Quebec, Quebec G1V 4G2, Canada

*S Supporting Information

ABSTRACT: Small molecule activators that directly modulate the activityof an enzyme are uncommon entities, and such activators had never yetbeen identified for any 17β-hydroxysteroid dehydrogenase (17β-HSD). Wehereby report the fortuitous discovery of a steroid derivative that caused anup to 3-fold increase in the activity of 17β-HSD12. The stimulation ofestrone to estradiol conversion has been characterized in intact andhomogenized stably transfected HEK-293 cells and has also been observedin T47D breast cancer cells. Structure−activity relationships closely linkedto the nature of the substituent on the [1,3]oxazinan-2-one ring of anestradiol derivative emerged from this study and may help in theidentification of a previously unsuspected endogenous activation of 17β-HSD12. This activator will therefore be a useful tool to study this relativelyunknown enzyme as well as the possible activation of other 17β-HSD familymembers.

A decade after its discovery, 17β-hydroxysteroid dehydro-genase type 12 (17β-HSD12) still remains relatively

unknown. It is one of the most recently identified isoenzymesof the 17β-HSD family, a group of key enzymes that are well-known to be involved in the last step of sex hormonebiosynthesis.1,2 This enzyme was however initially reported as3-ketoacyl-CoA reductase (KAR) for its essential role in fattyacid elongation cascade.3 It was later found to be able to reduceestrone (E1) in estradiol (E2) and was consequently renamed17β-HSD12.4 Since then, its physiological role remains a matterof debate. 17β-HSD12 appeared to be responsible for theincrease of potent estrogen biosynthesis in differentiatedadipocytes.5 On the other hand, some recent works with 17β-HSD12 knockout mice6 and cancer cells7,8 tend to show agreater involvement in fatty acid elongation. Both products,namely, E2 and long chain fatty acids, are known to be involvedin a variety of cancers, the former by the activation of estrogenreceptors (ER)2,9 and the later via further transformation intoeicosanoids.10 The expression of 17β-HSD12 has been linkedto poor prognoses of breast,7 ovary,11 and prostate12 cancersand is known to be stimulated by sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor related tofatty acid metabolism.13 Until now, however, no post-translational modulation of the enzyme had been brought toour attention.We recently evaluated the selectivity of certain enzyme

inhibitors developed in our laboratory by testing their 17β-HSD12 inhibition abilities in stably transfected HEK-293 cells.Surprisingly, a compound first designed as a 17β-HSD1inhibitor was found to more than double the E1 to E2conversion basal activity. This was an unexpected result,

considering that no molecule had been reported to turn on a17β-HSD nor any other dehydrogenases or steroidogenesisenzymes, as far as we know. The discovery of a small moleculeactivator that directly interacts with the enzyme to enhance itsactivity is a rather rare event as only 12 enzymes have well-known synthetic activators to date.14 The selective activation ofan enzyme activity with a small molecule may potentially offercrucial knowledge about its endogenous regulation and couldalso generate a great interest in therapeutic ends as well as invarious aspects of fundamental research.15

The existence of a 17β-HSD12 activator is interesting on itsown, considering how little is known about this essentialubiquitous enzyme.6,16 Such an activator could thereforebecome a useful tool, which is currently lacking, to study17β-HSD12. Moreover, it sheds light on an unsuspectedregulation pathway that could change the way we believe thiscrucial enzyme works. Thus, following the discovery of anactivation effect caused by a first small molecule, both theactivator and its effect were primarily characterized.

Screening for Stimulation of 17β-HSD12 Activity(Intact Cells). After an initial fortuitous observation ofenzymatic activity stimulation, available compounds synthe-sized in our laboratory with a closely related structure (Figure1) were screened for 17β-HSD12 inhibition/activation of[14C]-E1 to [14C]-E2 conversion in stably transfected HEK-293cells. These compounds (A1, A2, A3, B1, B2, B3, and B4) were

Received: September 1, 2013Accepted: June 2, 2014Published: June 2, 2014

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obtained by adapting a diversity oriented synthesis (DOS)methodology recently reported by our research group for thesynthesis of various fused steroidal azacycles.17 For thesynthesis of representative compounds A1 and A3, 3-methoxymethyl-O-estrone was submitted to an aldol con-densation with 3-formyl-benzonitrile to give the intermediateenone (Scheme 1). The carbonyl of this conjugated ketone wasthen stereoselectively reduced with sodium borohydride to givethe 17β-OH derivative, which was treated with a solution of m-chloroperbenzoic acid in chloroform. After a separation of bothepoxide derivatives by chromatography, the major epoxide withan alpha stereochemistry was heated at a high temperature in amicrowave apparatus with an amine (ethylamine or butyl-amine) to afford the corresponding aminodiol intermediate.Finally, the cyclization between the secondary amine and theOH group using triphosgene followed by the hydrolysis of the3-methoxymethyl ether protecting group provided the finalcompounds A1 and A3. Each compound was purified bychromatography and characterized to confirm their chemicalstructure and purity (Supporting Information).As initially observed, cells treated with compound A1 (10

μM) showed an increase of 291% in E2 production when

compared to the level of cells treated with vehicle only (Figure2A). In this screening assay, compound 55,18 a known 17β-

HSD12 inhibitor, was used as control. None of the othercompounds stimulated or inhibited the enzyme activity at 10μM. As a preliminary structure−activity relationship (SAR)statement, the R2 hydrophobic side chain seems crucial to theactivity since A2 only differs from A1 by a 2-hydroxy-ethylchain in R2 instead of a butyl group. Also, the activating effect

Figure 1. Chemical structure of compounds tested for 17β-HSD12modulation (activation and inhibition).

Scheme 1. Chemical Synthesis of Compounds A1 and A3a

aReagents and conditions: (a) methoxymethylchloride, diisopropylethylamine (DIPEA), dichloromethane (DCM), rt; (b) 3-CN-benzaldehyde,KOH, EtOH, 100 °C; (c) NaBH4, MeOH, rt; (d) m-chloroperbenzoic acid, DCM, rt; (e) appropriate alkylamine, EtOH, microwave, 180 °C; (f)triphosgene, DIPEA, DCM, rt; (g) HCl 10% (v/v) in MeOH, 50 °C.

Figure 2. Modulation (activation or inhibition) of the transformationof [14C]-E1 to [14C]-E2 by 17β-HSD12 in transfected HEK-293 intactcells. (A) Screening of compounds 55, A1, A2, B1, B2, B3, and B4tested at 10 μM. Ctrl: vehicle only. (B) Stimulation of 17β-HSD12activity by compounds A3 (gray) and A1 (black). Results of oneexperiment performed in triplicate (mean ± standard deviation).

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of this chain is highly affected by R1 substitution on theestratriene core. This could explain the lack of stimulationobserved by compounds from the B series, particularly B1,which differs from A1 only by an unsaturation at position 15−16 (Δ15−16) instead of a 16α-hydroxy group on a saturated D-ring. From our first results, no stimulation of enzyme activitycould be imputed to R3 substitution, but B3 and B4 were theonly two tested compounds with other substitutions thancarboxamide and showed no activation just like theircarboxamide counterparts B2 and B1. Thus, a 16α-OH-estratriene core as well as an adequately positioned hydro-phobic chain on the E-ring seems favorable for the stimulationof E2 production by 17β-HSD12 stably overexpressed in HEK-293 cells.In order to confirm the activation effect of a hydrophobic R2

group, compound A3 was synthesized and tested for thetransformation of [14C]-E1 by 17β-HSD12 (Figure 2B). Thiscompound only differs from A1 by the replacement of a butylby an ethyl group in R2. The production of [14C]-E2 wasstimulated by a treatment with A3 at concentrations of 10 and20 μM, but not at 1 μM. This stimulation by A3 was, however,considerably lower than that by A1 (36% and 57% stimulationagainst 89% and 202% at 10 and 20 μM, respectively). Thisresult confirms that the activation effect of A1 is not an artifactand also importantly validates the significant role of thehydrophobic R2 side chain for the observed stimulation. Itwould also be interesting to test compounds with various R2hydrophobic substitutions and different R3 moieties for anaccurate SAR determination, which may lead to synthesis ofbetter synthetic activators or to the identification ofendogenous compounds susceptible to exert similar or betteractivation of 17β-HSD12.Characterization of the Activation Effect in HEK-293

Intact Cells and Cell-Free Assays. Since compound A1, as asteroid derivative, might interact with various targets other than17β-HSD12, further tests were needed to determine thepossible mode of action responsible for the observed increase ofE2 production. To verify the hypothesis of a receptor-mediatedenzymatic stimulation of activity and/or expression, A1 wastested at many concentrations in both intact cells and cell-freeassay.Activation assays with compound A1 in intact cells showed a

clear dose-dependent stimulation of E1 to E2 conversion(Figure 3A) in the range of concentrations tested. The maximalactivation was however not reached considering that higherdoses have cytotoxic effect. The EC50 (effective concentrationnecessary to stimulate 50% of the maximal activity) and themaximal activation of basal activity have consequently not beendetermined for A1. For cell-free assays, a microsomal extractwas isolated from a homogenate of HEK-293 cells stablyexpressing 17β-HSD12 as previously described.4,19 A dose-dependent stimulation of E1 to E2 conversion by microsomalextract has been shown for 2.5 to 10 μM A1 with the maximalactivity observed at around 20 μM (Figure 3B). An EC50 of 7.5μM and a maximal activation of 120% were evaluated for A1.To determine whether compound A1 interacts with 17β-HSD12 by an allosteric site, we calculated the ratio between thecompound A1 (S) concentrations at 90% and 10% of Vmax(S0.9/S0.1). If an enzyme follows Michaelis−Menten kinetics inwhich the substrate concentration is equal to Km at Vmax/2, theS0.9/S0.1 value will be equal to 81.20 From data obtained in cell-free assay (Figure 3B), we determined a S0.9/S0.1 ratio of 8.9,which value indicates that compound A1 activates the 17β-

HSD12 by an allosteric site. This ratio value also allowed us toestimate the number of interacting sites as 2.The hypothesis of any genomic, transcriptional, or

proliferative effect of compound A1 is overturned by theobserved activation in a cell-free assay, without enzymes otherthan 17β-HSD12 involved in E1 to E2 metabolism4 and withthe lack of transduction pathways.21 In fact, the doubledenzyme activity observed in as little as 1 h is fairly unlikely to bemediated by a receptor or any other enzyme than the 17β-HSD12 in a microsomal extract of stably transfected HEK-293cells. A direct post-translational activation of the 17β-HSD12 isthus the only plausible explanation for the results in the cell-free assay. Nonetheless, the consistently higher maximalstimulation in intact cells than in microsomal fraction mightpoint out that compound A1 has additional effect in intact cells.Microsomal extraction process, change of enzymatic environ-ment, assay conditions (different substrate concentration andtime), or other factors affecting enzyme activity are most likelyto be responsible for this difference as the effectiveconcentrations remain unchanged in intact cells and in cell-free assay. These similar EC50 values are consistent with theeffect of an enzyme activator, because neither the absence ofintact cells nor the lack of cytoplasmic element seems to affectthe potency of compound A1.

Activation of 17β-HSD12 in Intact T47D Cells. Toconfirm the activation effect in more physiological-likeconditions than the enzyme overexpressed in HEK-293 cells,activation assays were conducted in T47D cells (breast

Figure 3. Stimulation of [14C]-E1 to [14C]-E2 conversion bycompound A1. (A) Experiment performed in intact HEK-293 cellsstably expressing 17β-HSD12. (B) Experiment performed in micro-somal extract obtained from homogenized HEK-293 cells stablyexpressing 17β-HSD12. Each figure is the result of one experimentperformed in triplicate (mean ± standard deviation) and isrepresentative of two assays.

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adenocarcinoma). These cells are well-known to show a highendogenous expression of 17β-HSD12 as well as a weakerexpression of 17β-HSD1, 17β-HSD7, and ER, which are proneto interact with the measurement of E1 to E2 conversion.22

Prior to the assay, modulation of 17β-HSD activities that mayinterfere with the results was first assessed (Figure 4A) using

T47D cells for 17β-HSD1 and transfected HEK-293 cells forthe three other 17β-HSDs (types 2, 7, and 12). Compound A1showed a good selectivity with 17β-HSD12 stimulation (235%)and a weak 17β-HSD1 inhibition (50%) at 10 μM only, whileno modulation of 17β-HSD2 and 17β-HSD7 was observed atboth 1 and 10 μM. It should be noted that the 17β-HSD1 is byfar the most efficient enzyme and is considered as being mainlyresponsible for E1 to E2 conversion.8,22 In fact, T47D cells areoften used to assess 17β-HSD1 inhibition as the activities of17β-HSD7 and 17β-HSD12 are not detectable in normalconditions.8,22 In order to detect any modulation of 17β-HSD12 activity, the 17β-HSD1 was totally inactivated with 1μM PBRM, a selective and irreversible inhibitor reported tohave a long-acting effect.23,24 No inhibitor of 17β-HSD2 and17β-HSD7 was used considering the lack of enzyme expressionand activity, respectively, in the T47D cells.22 However, thecells had to be cultured at their maximal density to avoid anyeffect through estrogenic stimulation caused by ER activation.

The transformation of E1 to E2 by the T47D cells increasedconsiderably from 0 to 72 h under all the conditions reported inFigure 4B. The rate of transformation was enhanced in aconcentration-dependent manner for cells treated with 5, 10,and 20 μM compound A1, which showed higher trans-formation values than control after 24, 72, and 120 h. At 1 μM,a small difference was observed only after 120 h. E1 to E2conversion appeared to reach a plateau after 72 h at allconcentrations except at 20 μM. In fact, no more E2 appearedto be produced from 72 to 120 h at 0 μM (27% vs 26%), 1 μM(28% vs 29%), 5 μM (32% vs 34%), and 10 μM (38% vs 41%)compound A1, whereas a small increase was observed at 20 μM(47% vs 55%). Such lowered and/or leveled off transformationafter 72 h is not surprising considering that at this point cellsare confluent in culture dishes for 5 days without a mediumchange. It should be noted that the observed production of E2by the 17β-HSD12 is very weak and the activity would not havebeen observed if the 17β-HSD1 was effective. Without the useof a 17β-HSD1 inhibitor, all of the substrate would have beentransformed22 in only a few hours, while the effect of 17β-HSD12 needs days to be adequately observed.The previous experiments thus confirmed the activation

potential of compound A1 in a breast cancer cell line. Suchactivation of 17β-HSD12 confirms the existence of anendogenous activation of the enzyme. Indeed, the activatorwe synthesized is most likely to mimic an endogenousactivation factor, as is the case for already characterized enzymeactivators.15 This possible modulation of 17β-HSD12, asobserved for the transformation of E1 to E2, could alsostimulate, inhibit, or have no effect on fatty acid elongation. Itwas however impossible to test this potential activity of 17β-HSD12 because of the actual lack of suitable substrates andassays. We do not even know if both enzyme activities arecatalyzed by the same catalytic site and how compound A1could stimulate these two activities. These two issues should beaddressed for a better understanding of the observed activationand its implications.For now, compound A1 may become a valuable tool to

investigate the probable endogenous activation of 17β-HSD12.At this time, we have found no therapeutic interest in activatingthis enzyme considering its relatively unknown role incancers7,12,22 and the fact that no physiological issue causedby 17β-HSD12 deficiency and/or lack of activity has yet beenreported in humans, as far as we know. Moreover, thetransformation catalyzed by this enzyme is not a rate-limitingstep in the production of essential long chain fatty acids, so itsactivation might have little or no effect25,26 and a significantphysiological involvement in E2 production remains uncertain.However, knowledge about the stimulation of 17β-HSD12activity might lead to insight in the development of potentinhibitors that could be interesting for the treatment of somecancers and inflammatory diseases.27 Additionally, this firstexample of a 17β-HSD activator gives rise to the possibleidentification of other 17β-HSD activators that could be ofgreat interest to treat, prevent, and/or study many diseases suchas cancer,1,2,27 endometriosis,28 osteoporosis,29 or disorders insexual development (formerly named pseudohermaphrod-ism)30 for instance.In summary, we report the identification of the first known

activator of a 17β-HSD family member. Thus, treatment withcompound A1 has produced stimulation of 17β-HSD12 activity(transformation of E1 to E2) in intact and homogenized stablytransfected HEK-293 cells as well as in intact T47D breast

Figure 4. (A) Evaluation of compound A1’s selectivity at 1 μM (gray)and 10 μM (black) for the transformation of [14C]-E1 by 17β-HSD1,17β-HSD7, and 17β-HSD12 and for the transformation of [14C]-E2 by17β-HSD2. T47D cells were used for 17β-HSD1 assays andtransfected HEK-293 cells for the other enzymes. (B) Stimulationby compound A1 of [14C]-E1 to [14C]-E2 transformation in T47Dcells cultured at confluence and treated with a potent 17β-HSD1inhibitor (PBRM). These are the results of one experiment in triplicate(mean ± standard deviation) representative of two assays.

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cancer cells. The exact mechanism of action is not yet known,but our results using a microsomal preparation suggest anallosteric binding of compound A1 to 17β-HSD12. Thisactivator could be an insightful tool to further study this littleknown enzyme and its modulation.

■ METHODSCells and Methods. T47D cells were obtained from the American

Type Culture Collection (ATCC), and HEK-293 cells stablytransfected with 17β-HSD12, 17β-HSD7, or 17β-HSD2 were kindlyprovided by Dr. Van Luu-The (CHUQ-CHUL Research Center,Quebec, Canada). Cell culture was conducted as previouslydescribed.31 The final concentration of dimethyl sulfoxide (DMSO)and ethanol in the culture medium was adjusted for each assay and didnot exceed 0.5% (v/v) together. [14C]-Radiolabeled substrate inethanol solution, namely, [14C]-E1 and [14C]-E2 (both from AmericanRadiolabeled Chemicals, Inc.), were used to assess enzyme activities.Steroids were extracted from medium with diethyl ether, separated bythin layer chromatography (plates from EMD Chemicals Inc.) withtoluene/acetone (4:1), and finally quantified using Storm 860 system(Molecular Dynamics) as described.32

Modulation of 17β-HSD12 in HEK-293 Cells. HEK-293 cellswere plated at 3 × 105 cells/well in 24-well culture dishes in MEMmedium as reported,31 but complete steroid-deprived medium wasobtained by using 10% (w/v) charcoal-stripped medium FBS (WisentInc.) instead of 10% FBS. After 48 h of incubation, cells were treatedby adding a DMSO solution of desired compound (0, 1, 10, and/or 20μM) and 60 nM [14C]-E1. For EC50 determination, final compoundconcentrations of 10 nM to 20 μM were tested. Culture medium wasremoved 24 h later for further quantification of radiolabeled steroids(E1 and E2).Modulation of 17β-HSD7 in HEK-293 Cells. HEK-293 cells

were plated in MEM complete steroid-deprived medium as above at adensity of 1.5 × 103 cells/well in 24-well culture dishes. After 48 h ofincubation, cells were treated with a DMSO solution of compound A1(0, 1, and 10 μM) and [14C]-E1 (60 nM). Culture medium wasremoved 18 h later for further quantification of radiolabeled steroids(E1 and E2).Modulation of 17β-HSD2 in HEK-293 Cells. HEK-293 cells

were plated in MEM complete steroid-deprived medium as above at adensity of 5 × 103 cells/well in 24-well culture dishes. After 48 h ofincubation, cells were treated by adding a DMSO solution ofcompound A1 and 60 nM of [14C]-E2. Culture medium was removed1 h later for further quantification of radiolabeled steroids (E1 andE2).Cell-Free Assay. Homogenates of HEK-293 cells expressing 17β-

HSD124 were obtained as previously reported19 except that cells werelysed by five freeze/thaw (−80 °C/4 °C) cycles and pipetting (up anddown) between each cycle instead of sonifying. Cell lysates werecentrifuged at 10000g, and the obtained supernatant was centrifugedagain at 100000g to isolate the microsomal fraction. The microsomalfraction was suspended in Tris buffer containing 20% (v/v) glyceroland BSA (0.2 mg/mL), and a sample of 100 μL was added to a testtube. The resulting suspension was treated with [14C]-E1 (10 nM) anda DMSO solution of compound A1 (final concentration of 0, 2.5, 5,10, 20, or 40 μM) in a total volume of 1 mL. After 1 h of incubation at37 °C with shaking, the tubes were immediately cooled down in ice,and the steroids (E1 and E2) were extracted for quantification.Inhibition of 17β-HSD1 in T47D Cells. T47D cells were plated

in RPMI complete steroid-deprived medium at a density of 3 × 103

cells/well in 24-well culture dishes as already described.24 After 48 h ofincubation, cells were treated by adding a DMSO solution of desiredcompound (0, 1, or 10 μM) and [14C]-E1 (60 nM). Culture mediumwas removed 24 h later for further quantification of radiolabeledsteroids (E1 and E2).Activity of 17β-HSD12 in T47D Cells. T47D cells were plated in

RPMI complete steroid-deprived medium at a density of 2 × 105 cells/well in 24-well culture dishes. After 24 h of incubation, DMSOsolution of 17β-HSD1 inhibitor PBRM (compound 23b in ref 23) was

added to the wells to reach a final concentration of 1 μM. After asecond 24 h incubation, cell medium was removed and replaced by 1mL of RPMI complete steroid-deprived medium containing 1 μMPBRM and 0, 1, 5, 10, or 20 μM compound A1 both in DMSOsolution. [14C]-E1 (60 nM final) was then added in the wells. Culturemedium was removed after 0, 24, 72, or 120 h for furtherquantification of radiolabeled steroids (E1 and E2).

Data Analysis. Both “Allosteric sigmoidal” and “log (agonist) vsresponse Variable slope” fits were performed using GraphPadPrism version 5.00 for Windows (GraphPad Software, www.graphpad.com) to analyze dose−response assays in intact cells and microsomalpreparation. Figure 3 panels A and B were drawn with the first fit,while EC50 was evaluated using the latter.

■ ASSOCIATED CONTENT*S Supporting InformationDetails of the chemical synthesis and characterization ofcompounds A1, A3 and intermediate compounds. This materialis available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: donald.poirier@crchul.ulaval.ca.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by a grant from the CanadianInstitutes of Health Research (grant number MOP43994) toD.P., an FRQS fellowship to A.T., and a CREMOGHfellowship to A.T. We would like to thank V. Luu-The(CHUQ-CHUL Research Center) for providing transfectedHEK-293 cells and M. Harvey for careful reading of themanuscript.

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dx.doi.org/10.1021/cb500109e | ACS Chem. Biol. 2014, 9, 1668−16731673