inhibition of dihydroorotate dehydrogenase activity by brequinar … · [cancer research 52....

[CANCER RESEARCH 52. .1521-3527. July 1. I992|

Inhibition of Dihydroorotate Dehydrogenase Activity by Brequinar SodiumShih-Fong Chen,1 Frank W. Perrella, Davette L. Behrens, and Lisa M. Papp

Cancer Research, Pharmaceutical Research, Drug Discovery Research, The Du Pont Merck Pharmaceutical Company, Glenolden Laboratory, Glenolden, Pennsylvania19036

ABSTRACT

The novel anticancer drug candidate brequinar sodium (DuP 785, NSC368390, 6-nuoro-2-(2'-fluoro-l,l'-biphenyl-4-yl)-3-methyl-4-quinoline-

carboxylic acid sodium salt) was shown previously to be an inhibitor ofdihydroorotate dehydrogenase, the fourth enzyme of the de novo pyrim-idine biosynthetic pathway. Brequinar sodium inhibits the activity of thisenzyme isolated from mammalian sources only but not those formsisolated from yeast or bacteria, which also use ubiquinone as the cofactor.Brequinar sodium also does not inhibit the activity of a soluble Zymo-bacterium oroticum dihydroorotate dehydrogenase which uses NAD'1' as

a cofactor. Brequinar sodium inhibits LI 210 dihydroorotate dehydrogenase with mixed inhibition kinetics with respect to either the substrate(dihydroorotate) or the cofactor (ubiquinone QÂ«)with A,' values in the

5-8 mi range. Our results suggest that brequinar sodium inhibits dihydroorotate dehydrogenase by binding to the enzyme at a unique site thatis distinct from the dihydroorotate or the ubiquinone-binding site. Thisbinding site appears to be unique to the mammalian enzyme, becausebrequinar sodium does not inhibit the yeast, EscherÃ¬chiacoli, or Z.oroticum forms of the enzyme.

INTRODUCTION

The novel anticancer agent brequinar sodium (DuP 785, NSC368390, 6-fluoro-2-(2'-fluoro-l,l'-biphenyl-4-yl)-3-methyl-4-

quinoline carboxylic acid sodium salt, Fig. 1) inhibits thegrowth of a broad spectrum of human solid tumors implantedin nude mice (1). Because of its activity against experimentaltumors, brequinar sodium was selected for further developmentand is now in phase 2 clinical trials. It has previously beenshown that brequinar sodium exerts its tumoricidal action byinhibiting the activity of DHO-DHase,2 the fourth enzyme in

the de novo pyrimidine biosynthetic pathway (2, 3).The finding that brequinar sodium inhibits the activity of

DHO-DHase is interesting since its structure does not resembleeither the substrate or the cofactor involved in the enzymaticreaction (Fig. 1). We have previously identified several important regions of the molecule that are essential for inhibiting theenzyme activity (4). To understand the nature of the interactionof brequinar sodium with its target, enzyme inhibition kineticsand the specificity of the inhibition of DHO-DHase fromvarious sources by brequinar sodium were studied. The inhibition kinetics of two known inhibitors of this enzyme, dichlo-i dally 1lawsone (5) and orotate (6), were also studied (Fig. 1).

Our results show that brequinar sodium inhibits the mammalian but not the nonmammalian DHO-DHases. A hypothetical model of the enzyme-brequinar sodium interaction is presented. Portions of this work have been presented in preliminaryform (7).

Received 8/12/91; accepted 4/23/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' To whom requests for reprints should be addressed, at Institute for Drug

Development, Cancer Therapy and Research Center, 8122 Datapoint Drive, Suite700, San Antonio. TX 78229.

! The abbreviations used are: DHO-DHase, dihydroorotate dehydrogenase, EC1.3.3.1; DCL, dichloroallyl lawsone; DCIP, 2,6-dichlorophenolindolphenol.

MATERIALS AND METHODS

Chemicals. Brequinar sodium was synthesized by the MedicinalChemistry Section, Pharmaceuticals Division, Du Pont Medical Products Department. Dichloroallyl lawsone was provided by the DrugDevelopment Branch of the National Cancer Institute, Bethesda, MD.DHO-DHase (Zymobacterium oroticum) and all other chemicals werepurchased from Sigma Chemical Company, St. Louis, MO. [carboxyl-14C]Orotic acid (specific activity, 60 mCi/mmol) and Biofluor were

purchased from New England Nuclear Research Products, Boston,MA. L-[cflrftoArv/-1'1C]Dihydroorotatewas synthesized as previously de

scribed (2). Whatman DE81 Chromatographie paper was purchasedfrom VWR Scientific, Philadelphia, PA.

Preparation of DHO-DHase from LI 210 Cells. L1210 cells harvestedfrom the peritoneal cavity of CD2F1 mice were washed twice with 0.9%NaCl and homogenized as described previously (2). The isolated intactmitochondria were then solubili/ed with Lubrol PX (1 mg protein:0.3mg Lubrol PX) and stored at â€”¿�80Â°C.The frozen enzyme was stable for

>1 year. The protein concentration was determined by the method ofBradford (8).

Preparation of Crude Homogenate of Tumor Cells. B16 murine melanoma, HL-60 human promyelocytic leukemia, and RPMI 7272 humanmelanoma cells were grown in RPMI 1640 medium supplemented with10% fetal bovine serum. Cells (1-2 x 10s) were harvested and resus-pended in 1 ml of 67 ITIMTris-HCl (pH 7.4) containing 1 itiM phenyl-methylsulfonyl fluoride. The cell suspension was sonicated at 4"C for

15 s. The crude homogenate was then solubilized in Lubrol PX (1 mgprotein:0.3 mg Lubrol PX) and stored at -80Â°C.

Preparation of DHO-DHase from Mouse Liver and Spleen. Mouseliver DHO-DHase was partially purified from mitochondria as reportedfor rat liver (9) and solubilized in Lubrol PX (1 mg protein:0.3 mgLubrol PX). Mouse spleen lymphocytes were prepared by gently homogenizing mouse spleens in 15 ml of RPMI 1640 medium supplemented with mercaptoethanol (4 Â¿il/litermedium) until only the capsulewas left. The cell suspensions were transferred into a centrifuge tubeand then centrifuged at 1000 x g for 20 min. The cell pellets wereresuspended in 30 ml of phosphate-buffered saline, and the cell suspensions were carefully layered on top of 15 ml of Ficoll and centrifugedat 1500 x g for 15 min. The lymphocytes were then removed andwashed with phosphate-buffered saline. A crude homogenate of lymphocytes was then prepared as described above for tumor cells.

Preparation of DHO-DHase from EscherÃ¬chiacoli. E. coli (K12 strain)from several isolated colonies growing on nutrient agar was inoculatedin 100 ml of Mueller-Hinton broth (10). The broth was incubatedovernight at 37Â°Cwith shaking. The broth containing E. coli was thendiluted 1:10 with Mueller-Hinton broth and further incubated at 37Â°C

until the absorbance of the culture reached about 0.4 at 540 nm. E. coliwere harvested by centrifugation, and the pellets were washed fourtimes with 70 HIMTris-HCl (pH 7.6). The pellets were frozen at -80Â°C

and thawed. The thawed E. coli pellets were suspended in 5 ml of cold70 HIMTris-HCl (pH 7.6) and sonicated intermittently for 15 s at 4'C.

The extent of cell disruption in the cell suspension was assessed underdark-field microscopy. When approximately 50% of the cells weredisrupted, the sonicated mixture was then centrifuged at 3000 x g untilthe supernatant fluid contained no intact cells. The supernatant fluidwas diluted with 1% Triton X-100 to give a final protein concentrationof 1 mg/ml in 70 m\i Tris-HCl (pH 7.6).

Enzyme Assay. The activity of DHO-DHase was determined by thedirect conversion of i.-[car/>0A:>7-'4C]dihydroorotate to [carboxyl-'*C]

orotate. Unless otherwise indicated, the standard reaction mixturecontained 67 HIM Tris-HCl buffer (pH 7.4), 5 m\i KCN, 600 MMubiquinone Qft, and Lubrol PX-solubilized DHO-DHase with or with-

3521

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INHIBITION OF DHO-DHase BY BREQUINAR SODIUM

Lbiquinono Qh(n = 6)

Fig. 1. Chemical structure of brequinar sodium and dichloroallyl lawsone andthe reaction catalyzed by dihydroorotate dehydrogenase.

out inhibitors in a total volume of 160 jil. The reaction mixture waspreincubated at room temperature for 30 min. The reaction was theninitiated by the addition of 40 Â¿ilof 5x concentrated L-[carboxyl-'''C]

dihydroorotate (10 Â¿<Mfinal concentration), and the rate of orotateformation was determined over a period of 8 min using the DE81Chromatographie system described earlier (2). To determine the cofac-tor requirement of the Lubrol PX-solubilized DHO-DHase from LI 210cells, mouse liver, and E. coli, ubiquinone was substituted with a suitableconcentration of other electron acceptors in the reaction mixture. Whenintact mitochondria were used in the reaction, mitochondria (15-20 Â¿igprotein) were incubated in a reaction mixture containing SO HIM4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid buffer (pH 6.8), 10%glycerol, and 2 imi dithiothreitol with or without inhibitors at roomtemperature for various periods of time before the addition of L-[carfto.r}'/-'4C]dihydroorotate. The rate of orotate formation was meas

ured as described above.Data Analysis. The rate law models for competitive, uncompetitive,

mixed inhibition, ping-pong bi bi, and random and ordered sequentialbi bi kinetics were "fit" to the enzyme kinetic data using the EZ-FIT

computer program for nonlinear regression analysis. This programadjusts the model parameters so that the sum of squares of the residualsbetween the data and the model are minimized (11). The identificationof the "best-fit" model among several rival models is determined, in

part, by using the criterion of Akaike for weighted and nonweighteddata. The Akaike criterion takes into consideration the number ofmodel parameters when determining the goodness of fit of rival models.The final identification of the best-fit model is based on several criteria(11): (a) the minimized Akaike test, (Â¿>)the t statistic of the fittedkinetic parameters at the 0.05 level of significance, (c) the number ofoutlying data points, and (d) the "Runs" test of residuals at the 0.05

level of significance to evaluate randomness of the residuals; an indication of whether the experimental data departs systematically fromthe "fitted" curve.

The initial velocity of orotate formation in the presence or absenceof inhibitors was estimated by nonlinear regression analysis of progresscurves using the integrated form of Michaelis-Menten equation usingEZ-FIT (11).

Three basic types of enzyme inhibition exist: competitive, mixedinhibition, and uncompetitive. To determine the type of inhibitionkinetics, the initial velocities were determined as described above with

the addition of different fixed concentrations of the inhibitor in thereaction mixture. The initial velocity data of orotate formation werethen fitted simultaneously to the following equations to determine thetype of inhibition and the inhibition constants.

Competitive inhibition:

i +

Uncompetitive inhibition:

+i/KÂ¡)

1 + (KJS) + (1/JQ

Mixed inhibition:

(KJS)*(\ + I/KÂ»)+ 1 +

Inhibitors are regarded as competitive if nonlinear regression analysisof the untransformed data produces a best fit of the competitive model.For visual purposes, double-reciprocal transformation and plotting ofthe data produces plots on which only the slope of the reciprocal plotis affected and not the intercept. Reciprocal plots of the data constructedusing different inhibitor concentrations produce regression lines thatcross on the vertical axis and are consistent with the elimination of theinhibitory effect as the variable substrate is saturating.

Inhibitors are regarded as uncompetitive if nonlinear regressionanalysis of the untransformed data produces a best-fit of the uncompetitive model. Double-reciprocal transformation and plotting of thedata produces plots on which only the intercept of the reciprocal plotis affected and not the slope. Reciprocal plots constructed using different inhibitor concentrations produce a pattern of parallel regressionlines.

Inhibitors are regarded as mixed inhibitory or noncompetitive ifnonlinear regression analysis of the untransformed data produces abest-fit of the mixed inhibition model. Double-reciprocal transformation and plotting of the data produces plots on which both the slopeand intercept of the reciprocal are increased. Reciprocal plots constructed using different inhibitor concentrations produce a pattern ofregression lines that (a) cross anywhere to the left of the vertical axisand (b) are either above, below, or on the horizontal axis. The termmixed inhibition is used here to refer to all classes of noncompetitiveinhibition for which both the slopes and the intercepts of doublereciprocal plots are increased by the inhibitor.

RESULTS

Inhibition of DHO-DHase Activity in Intact and Lubrol PX-Solubilized LI 210 Mitochondria by Brequinar Sodium. The activity of LI 210 DHO-DHase can be measured in both intactmitochondria and in detergent (Lubrol PX) solubilized mitochondria. No exogenous cofactors were needed to measure theenzyme activity when intact mitochondria were used. Brequinarsodium inhibited the intact mitochondria! DHO-DHase activityin a time-dependent manner. The inhibition was not due to theinstability of the enzyme in isolated mitochondria, since theactivity in control experiments was only affected slightly (Fig.2). Maximal inhibition was achieved when LI 210 mitochondriawere preincubated with brequinar sodium over a long period oftime (approximately 2 h). When intact mitochondria weresolubilized with Lubrol PX, little enzyme activity could bedetected unless exogenous cofactors were added (Table 1).Several ubiquinones, potassium ferricyanide, and DCIP can beused as the exogenous cofactors of the LI 2 10 DHO-DHase.Because ubiquinone Q6 was reported as the natural cofactor ofmammalian DHO-DHase (12), it was used in our standardassay. The inhibition of enzyme activity by brequinar sodium

3522




100O

10

0 15 30 45 60 75 90 105 120 135

Time (Min)

Fig. 2. Time-dependent inhibition of LI 210 DHO-DHase in intact mitochondria. Intact L1210 mitochondria were incubated at room temperature in thepresence of 0 (D) and 100 (â€¢)nM brequinar sodium for the indicated timeintervals. The remaining DHO-DHase activity was measured as described in"Materials and Methods."

Table 1 Utilization of various electron acceptors as cofactor ofLI210 andmouse liver DHO-DHase

The activity of Lubrol PX-solubilized DHO-DHase was determined by thedirect conversion of L-[coreo.r>7-'4C]dihydroorotate to [caroo.t)'/-'<C]orolate asdescribed in "Materials and Methods" except that the ubiquinone was substituted

with the appropriate electron acceptors.

LI2IO" Mouse liver" Bovineliver*

Electron acceptorRelative Relative Relative

Activity' activity Activity' activity activity

UbiquinoneQÂ«p-BenzoquinoneUbiquinone

QoUbiquinoneQ,UbiquinoneQ,Ubiquinone(.>,.MenadioneVitamin

K1DCIPPotassium

ferricyanidePhenazinemethosulfateCytochromeCOxygen11.451.312.829.194.664.480.50.313.922.060.190.150.25|100]1024.680.240.739.14.32.334.217.91.41.12.00.48NT*0.030.560.170.1NTNT0.10.09NTNT0.03[100]NT6.011734.621.7NTNT21.719.4NTNT7.1|100]152097NTNT19NT144NTNT0

Â°Enzyme source is from the mitochondria.'' Published results cited in Ref. 19.' Activity is nmol/min/mg protein.d NT, not tested.

UM of brequinar sodium (a concentration which completelyinhibits the enzyme activity) and was subtracted from each ofthe data points before data analysis. This method provided amore accurate determination of the initial velocity than thatapproximated by fitting the data to a straight line.

Initial Velocity Pattern of Dihydroorotate Oxidation. Theinitial velocity pattern of dihydroorotate oxidation was studiedto verify the reaction mechanism of DHO-DHase. The initialvelocities of orotate formation were obtained by varying theconcentration of one substrate at several different fixed concentrations of the other substrate. The data were plotted in double-reciprocal form (Fig. 4). Parallel straight lines were obtainedwhether dihydroorotate or ubiquinone Q6 was used as thevariable substrate. This initial velocity pattern suggests that theLI210 DHO-DHase reaction follows a ping-pong mechanism(13). The analysis of the data using random and ordered sequential models gave poorer fits.

Kinetics of Inhibition of I 12III DHO-DHase Activity by Brequinar Sodium, Dichloroallyl Lawsone, and Orotate. Inhibitionof LI210 DHO-DHase was studied by varying either the firstsubstrate (dihydroorotate) or the second substrate (ubiquinone)concentration at various inhibitor concentrations. An exampleof the inhibition of DHO-DHase activity by brequinar sodiumwith a fixed ubiquinone concentration (60 UM) at variabledihydroorotate concentrations (1.67-10 Ã•Ã•M)is shown in Fig. 5.The initial velocity of the orotate formation was estimated asdescribed in "Materials and Methods." The reaction velocity

data were then fitted using the nonlinear least squares methodof EZ-FIT (11) to three kinetic models (competitive, uncom-petitive, and mixed inhibition) to determine the type of inhibition by their goodness of fit. This method of analysis avoids theerrors that can arise from double-reciprocal data analysis andprovides an estimate of the standard errors of the kineticconstants (/Â£"â€žâ€žFmax,and K{). It was determined that a mixed

inhibition model fit the data best. The data were then plottedas the typical Lineweaver-Burk (double-reciprocal) plot forvisual evaluation.

o

was no longer time dependent when using Lubrol PX-solubilized DHO-DHase. Under these conditions, maximal inhibitionwas achieved almost instantly. To be certain that the inhibitionwas complete, a 30 min preincubation of Lubrol PX-solubilizedenzyme with inhibitors was used before adding the substrate toinitiate the enzymatic reaction in our assay.

Initial Velocity Determination of Orotate Formation. The timecourse of orotate formation is shown in Fig. 3. The rate oforotate formation was not linear during the time course (8 min)of the experiment. To estimate the initial velocity of the reaction, the data were fitted to the integrated form of the Michaelis-Menton equation (11). The background radioactivity at eachsubstrate concentration was determined in the presence of 25

3523

Â£

Time (Min)Fig. 3. The time course of orotate formation. Lubrol PX-solubilized LI210

mitochondria were incubated with graded concentrations of L-{carhoxyl-'4C]dihydroorotate. 1.67 (â€¢),2.0 (O), 2.5 (â€¢),3.3 (D). 5.0 (A), and 10 (A) >iM.andthe rate of orotate formation was determined as described in "Materials andMethods."



INHIBITION OF DHO-DHase BY BREQL'INAR SODIUM

The inhibition of LI210 DHO-DHase by brequinar sodium,dichloroallyl lawsone, and orotate is summarized in Table 2.Brequinar sodium and dichloroallyl lawsone inhibited DHO-DHase with mixed inhibition kinetics when measured at variable concentrations of dihydroorotate (1.67-10 UM)and a constant concentration of ubiquinone (60 MM).In contrast, orotateinhibited DHO-DHase competitively when the the same protocol was used. Brequinar sodium and dichloroallyl lawsonealso inhibited the activity of DHO-DHase with mixed inhibitionkinetics when variable concentrations of ubiquinone (10-60MM)were used at a fixed concentration of dihydroorotate (10MM). Under these conditions, orotate inhibited the enzymeuncompetitively. The Aâ„¢of the substrate (dihydroorotate) was1.4 Â±0.2 MM(mean Â±SD of 6 determinations); the Km of thecofactor (ubiquinone) was 40.3 Â±11.8 MM(mean Â±SD of 7determinations).

Inhibition of Various DHO-DHases by Brequinar Sodium andDichloroallyl Lawsone. Brequinar sodium and dichloroallyl lawsone at 100 n\i also inhibited mouse liver DHO-DHase; thedegree of inhibition obtained was similar to that observed usingthe LI210 enzyme (Table 3). However, brequinar sodium anddichloroallyl lawsone at 50 MM(500-fold higher concentration)did not inhibit the enzyme isolated from baker's yeast (Saccha-

romyces cerevisiae) and bacteria (E. coli). Both the yeast and E.coli enzymes utilize ubiquinone Q6 as the cofactor. In addition,the commercially available soluble enzyme (Z. oroticum), whichis known to use NAD* but not ubiquinone as the cofactor, was

not inhibited by 50 MMof brequinar sodium or dichloroallyllawsone. The results of the inhibition of DHO-DHase by dichloroallyl lawsone are in agreement with those reported byBennett et al. (5).

The activities of DHO-DHase in the crude homogenates ofvarious normal tissues and tumor cells were determined (Table4). The activities of enzyme obtained from tumor cells werehigher than those from normal cells when the activities wereexpressed as nmol/min/109 cells. The differences in the enzyme

activities between the tumor cells and normal tissue were muchless if they were normalized to nmol/min/mg protein, because

2o.ec

01o.

â€”¿�"0

o.II

-1.0

1/DHO (1/jaM)

Fig. 5. Determination of inhibition kinetics of brequinar sodium with LubrolPX-solubilized LI 210 DHO-DHase. Double-reciprocal plots of velocity (>')versusdihydroorotate concentration at a fixed ubiquinone (60 ,.\n and various fixedconcentrations of brequinar sodium: 0 (O). 5 (â€¢).10 (D). 15 (â€¢),and 20 (A) n\i.Velocity is the pmol of orotate formed/min/mg protein.

the tumor cells contained higher protein concentrations. TheDHO-DHases in the crude homogenates of both the normaland the tumor cells were inhibited by 100 HMbrequinar sodiumto a similar extent. These results indicate that brequinar sodiumdoes not preferentially inhibit DHO-DHase isolated from normal versus tumor cells.

DISCUSSION

We have demonstrated that the novel anticancer agent, brequinar sodium, is a potent inhibitor of mammalian DHO-

"= 4-

â€¢¿�3 3-

x

8o>- 11

(A)

egvCL.

o

O.

4-

^ 'I ~

O 2-

1-

20 40 60 80 100 120 140 0 10 15 20 25 30 35

1/Dihydroorotate (1/u.M x 1000) 1/Ubiquinone (1/u.M x 1000)Fig. 4. Initial velocity pattern for dihydroorotate oxidation. In I. orotate formation was determined at 6 fixed concentrations of ubiquinone (.)â€ž.The reciprocal

velocities are plotted as a function of the dihydroorotate concentration. The concentrations of ubiquinone used Â»ere:33 (â€¢).40 (O), 50 (â€¢).67 (G). 100 (A), and 200(A) tiM. In B, orotate formation was determined at 6 fixed concentrations of dihydroorotate. The reciprocal velocities are plotted as a function of the ubiquinoneconcentration. The concentrations of dihydroorotate used were: 8 (â€¢),10 (O), 12.5 (â€¢),16.6 (D), 25 (A), and 50 (A) JIM.

3524




DHase. DHO-DHase catalyzes the oxidation of dihydroorotateto orotate (Fig. 1). This reaction is the single redox step in thepyrimidine de novo biosynthetic pathway. Several DHO-DHases from different species are known. The major differencein these enzyme forms appears to be the cofactor requirement.The commercially available DHO-DHase isolated from Z. or-oticum is a soluble enzyme that uses NAD+ as cofactor. Pro-

karyotic DHO-DHase is a membrane-bound enzyme which usesubiquinone Q,<>as the natural cofactor (10). Cytosolic enzymeshave been isolated from the parasitic protozoan Crithidia fas-

ciculata and Trypanosoma brucei (14,15); both proteins containflavin as the only redox-active cofactor and utilize molecularoxygen as the cosubstrate electron acceptor. Mammalian DHO-

DHase and the enzyme isolated from Neurospora crassa aremitochondrial enzymes which are membrane associated (6, 16).These enzymes appear to be linked functionally and, perhaps,structurally to the electron transport system. The N. crassaenzyme is an iron-containing flavoprotein that uses long-chainquiÃ±onesas cosubstrate electron acceptors (17, 18). The bovineliver mitochondrial DHO-DHase has optimal activity withubiquinone as cosubstrate and contains flavin mononucleotideand iron (19). Our study of the cofactor requirement demonstrates that ubiquinone Q6 was the best cofactor for both theLI210 and mouse liver enzymes; the cofactor requirement ofthe LI210 and mouse liver enzymes, therefore, resembles thatof the bovine liver enzyme (19).

The DHO-DHase-catalyzed reaction has been reported tofollow the ping-pong mechanism; dihydroorotate binding precedes ubiquinone binding (20, 21). Our studies of the initialvelocity pattern for dihydroorotate oxidation by the LI210enzyme yields parallel lines in the double-reciprocal plots,indicative of a ping-pong mechanism. However, our inhibitionstudies showed that orotate was a competitive inhibitor withrespect to dihydroorotate and an uncompetitive inhibitor withrespect to ubiquinone. These results are in contrast to theclassical ping-pong mechanism (13). In the classical ping-pongmechanism, the first substrate (dihydroorotate) and the secondproduct (ubiquinol) would both combine with the same form ofthe enzyme, whereas orotate and ubiquinone would combinewith an alternative form. The matching pairs of substrate andproduct in the classical mechanism produce a noncompetitivetype inhibition pattern, and it is presumed that one binding site

Table 3 Inhibition of DHO-DHase isolated from various sources by brequinarsodium and dichloroallyl lawsone

The enzyme activity of DHO-DHase was measured in the absence and thepresence of 100 nM or 50 MMof brequinar sodium as described in "Materials andMethods."

EnzymesourceL1210"Mouse

liver"S.

cerevisiae"E.

coli'Z.

OTOtÃ¬CIUlfInhibitorBrequinar

sodiumDCLBrequinar

sodiumDCLBrequinar

sodiumDCLBrequinar

sodiumDCLBrequinar

sodiumDCLConcentrationlOOnMHill

n\llOOn.M100

n\i50MM50MM50

MM50MM50

MM50MM%

inhibition85807890000000

Â°Ubiquinone Q6 was used in the enzyme reaction.* NAD* was used in the enzyme reaction.

for the catalytic process exists in the enzyme for both substrates.As stated above, our data are not in agreement with this classicalping-pong mechanism.

Recently, using highly purified DHO-DHase from bovineliver mitochondria, HiÃ±esand Johnston (21) showed that thebovine enzyme may follow a nonclassical, two-site ping-pongmechanism. Orotate was also shown to be a competitive inhibitor with respect to dihydroorotate. In a nonclassical ping-pongmechanism, the two substrates may not resemble each otherchemically, and therefore, they may not bind to a single bindingsite but probably bind to two adjacent sites that transfer thereactants. This mechanism is typical of an enzyme that containstwo nonoverlapping and kinetically isolated substrate-bindingsites (21). A plausible explanation for our data concerningmammalian DHO-DHase is that its kinetics follow that of anonclassical, two-site model. The product inhibition patterns

using orotate as the inhibitor for the LI210 enzyme (Table 2)are similar to those reported for the bovine enzyme (21), suggesting that the L1210 DHO-DHase also follows a nonclassical,two-site ping-pong mechanism.

Unlike orotate, brequinar sodium inhibits the L1210 enzymewith mixed inhibition kinetics with respect to either dihydroorotate or ubiquinone. These results suggest that brequinar sodium may not compete directly at either the dihydroorotate- orthe ubiquinone-binding sites. Our finding that brequinar so-

Table 2 Inhibition of LI 210 DHO-DHase by brequinar sodium, dichloroallyl lawsone, and orotateLubrol PX-solubilized L1210 DHO-DHase was incubated with various inhibitor concentrations and with (a) 60 MMof ubiquinone Q6 and various dihydroorotate

concentrations (1.67-10 MM)or with (A) 10 MMdihydroorotate and various ubiquinone concentrations (10-60 MM).The residual DHO-DHase activity was determinedas described in "Materials and Methods."

Inhibitor

Variable substrate

Dihydroorotate Ubiquinone Q6

Brequinar sodium

Dichloroallyl lawsone (DCL)

Orotate

AÂ¡,(nM)K,2(nM)Kâ€ž(MM)

Pattern

A"Â¡,(nM)

Ka (n\i)Km (MM)

Pattern

A",(MM)A'm(MM)

KiuxPattern

4.9 Â±1.2Â° (7.0 Â±2.5)*

7.4 Â±0.9 (3.9 Â±0.5)1.4 Â±0.2 (1.9 Â±0.2)7.6 Â±0.4 (19.3 Â±0.8)

Mixed inhibition

2.2 Â±0.8 (2.7 + 0.7)3.0 + 0.5 (3.0 + 0.3)1.4 Â±0.3 (1.3 Â±0.2)8.0 + 0.4 (15.7 + 0.4)

Mixed inhibition

1.5 + 0.1 (1.1+0.1)1.3 Â±0.1 (1.2 Â±0.2)6.8 + 0.2 (16.6 + 0.7)

Competitive

6.7+ 1.8 (7.8 Â±1.8)5.7+ 1.4 (5.0 Â±1.0)

33.9 Â±5.7(45.1 +7.0)25.1 + 2.2(31.0 + 2.7)

Mixed inhibition

2.9 Â±0.4 (2.2 Â±0.4)3.7 Â±0.7 (2.2 Â±0.6)

35.4 Â±4.0(63.5 Â±9.1)32.7 Â±1.9 (38.9 Â±3.5)

Mixed inhibition

5.8 Â±0.4 (6.4 Â±0.4)45.6+ 4.0 (36.2 Â±3.5)37.5 Â±1.9 (28.8 Â±1.5)

Uncompetitive' Estimated value Â±SE of the fit.* Values within parentheses are the results of a separated experiment.' Vâ„¢*= nmol orotate formation/min/mg protein.

3525




Table 4 Inhibition of mammalian DHO-DHase activities by brequinar sodiumThe enzyme activity of DHO-DHase of the crude homogenates was measured in the absence and presence of 100 nM of brequinar sodium as described in "Materials

and Methods." The enzyme activity was expressed as nmol/min/mg protein or nmol/min/10' cells.

CellsL12IO

B16HL-60RPM1 7272SpleenSpleen + phytohemaglutinin"Spleen + lipopolysaccharide*Protein

(mg/10'cells)80.8

243.9113.9268.3

11.844.938.4nmol/min/mg

protein0.75

0.370.350.220.0850.1730.172nmol/min/109

cells62.1

79.856.358.6

1.18.55.8%

inhibition85.0

78.094.280.883.577.071.4

" Mice spleen lymphocytes were prepared as described in "Materials and Methods" and were incubated with 2 jjg/ml phytohemaglutinin in RPMI 1640 mediumat 37Â°Cfor 3 days.

* Mice spleen lymphocytes were prepared as described in "Materials and Methods" and were incubated with 0.1 /ig/ml lipopolysaccharide in RPMI 1640 mediumat 37Â°Cfor 3 days.

DHO OA

UBQL UBQ

Active Form

UBQ

Inactive Form

Fig. 6. Proposed model for the interaction of brequinar sodium with DHO-DHase. UBQ, ubiquinone; UBQL, ubiquinol; OA, orotate; B, brequinar sodium;FMN, flavin mononucleotide.

dium does not inhibit the S. cerevisiae or E. coli DHO-DHaseseven though they use dihydroorotate and ubiquinone as substrate and cofactor, respectively, provides further evidence thatbrequinar sodium may not compete directly with the substrate-binding site on DHO-DHase. These results suggest that thereis a unique binding site for brequinar sodium on the mammalianenzyme, which may not exist on the S. cerevisiae and E. colienzymes.

The potency of brequinar sodium for the inhibition of theLI210 enzyme is substantially greater than that reported forthe rat liver enzyme (3). This disparity in potency may be dueto the limited sensitivity of the high-performance liquid chro-matography method used in the rat liver studies (3). Using aradiolabeling assay, we found that brequinar sodium inhibitsthe activities of the enzymes isolated from mammalian normaltissues or tumor cells equally well. Dichloroallyl lawsone, astructural analogue of ubiquinone, was originally thought to bea respiratory poison and was shown later by Bennett et al. (5)to be a potent inhibitor of DHO-DHase. Using intact mitochondria from rat liver, these workers demonstrated that dich-loroallyl lawsone was an uncompetitive inhibitor of the enzymewith respect to dihydroorotate. Using Lubrol PX-solubilizedLI210 enzyme, we observed that dichloroallyl lawsone, similarly to brequinar sodium, inhibits DHO-DHase with mixedinhibition kinetics with respect to either dihydroorotate orubiquinone. Since Lubrol PX-solubilized L1210 DHO-DHaseis used in this study, we were unable to determine whether thedifferences in the inhibition patterns reported by us and byBennett et al. are due to the source or to the preparation of the

enzymes. However, dichloroallyl lawsone did not inhibit the S.cerevisiae and E. coli enzymes, suggesting that dichloroallyllawsone may also not bind directly to the ubiquinone-bindingsite.

In summary, the model that best fits our data is one in whichDHO-DHase follows a nonclassical two-site ping-pong mechanism; the substrate (dihydroorotate) and the cofactor (ubiquinone) binding sites are nonoverlapping and kinetically distinct(Fig. 6). These two sites are probably linked by an intramolecular electron transfer system involving flavin mononucleotideand perhaps iron or zinc. Brequinar sodium may bind to thiselectron transfer system and alter the conformation of thesubstrate and/or cofactor-binding sites. Further elucidation ofthe precise nature of the brequinar and DHO-DHase interactionmay depend on X-ray crystallographic studies.

ACKNOWLEDGMENTS

The authors are grateful to Dr. D. L. Dexter for encouraging thisinvestigation and for many suggestions and critically editing this man -uscipt. We also thanks Drs. M. Forbes, N. Ackerman, and R. C. Jacksonfor their support.

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1992;52:3521-3527. Cancer Res Shih-Fong Chen, Frank W. Perrella, Davette L. Behrens, et al. Brequinar SodiumInhibition of Dihydroorotate Dehydrogenase Activity by

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