Characterization of the Interaction of 2-[2-Nitro-4-(trifluoromethyl

with a specific activity of 54.8 mCi/mmol and a radiochemical purity of 98.7% was purchased from Amersham International plc (Buckinghamshire, U.K...
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Chem. Res. Toxicol. 1996, 9, 24-27

Characterization of the Interaction of 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione with Rat Hepatic 4-Hydroxyphenylpyruvate Dioxygenase Martin K. Ellis,*,† Alison C. Whitfield,† Lesley A. Gowans,† Tim R. Auton,† W. McLean Provan,† Edward A. Lock,† David L. Lee,‡ and Lewis L. Smith§ Zeneca Central Toxicology Laboratory, Alderley Park, Nr Macclesfield, Cheshire SK10 4TJ, U.K., and Zeneca Ag Products, Western Research Centre, Richmond, California 94804-002 Received June 13, 1995X

The synthetic β-triketones are a novel family of chemicals, developed as herbicides that have activity on grass and broadleaf weeds and are selective in corn. Toxicological evaluation of a number of these chemicals has established that they interfere with rat hepatic tyrosine catabolism in vivo by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD). This paper describes the kinetics of inhibition of rat hepatic HPPD in vitro by the representative β-triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (1). A marked inhibition of rat hepatic HPPD was observed when 1 was incubated with the enzyme for 3 min at 37 °C prior to the initiation of the enzyme reaction by the addition of substrate. In this system, a concentration of 200 nM 1 resulted in a >90% loss of HPPD activity, and an apparent IC50 was established at approximately 50 nM. The rate constant for the inactivation of HPPD by 1 was (1.5 ( 0.2) × 10-5 s-1 nM-1 as determined by progress curve data of oxygen consumed by HPPD with time. This inhibition is reversible in that the enzymeinhibitor complex slowly dissociates, with approximately 5.5 ( 0.6% of the enzyme activity being recovered by 6 h at 25 °C (t1/2, 25 °C, estimated at 101 ( 14 h). In short, our studies establish 1 to be a tight-binding inhibitor of rat hepatic HPPD in vitro. This inhibition is characterized by the rapid inactivation of HPPD by the formation of an enzyme-inhibitor complex that dissociates extremely slowly with recovery of enzyme activity.

Introduction The β-triketones, first reported by Penfold in 1920 (1), are a generic family of chemicals that have a triacylmethane [enol-tautomer; 3-(1-hydroxyethylidene)pentane2,4-dione] functionality incorporated within their structure. The naturally occurring β-triketones are classified into three groups: those derived from a phloroglucinol ring (2-4), a resorcinol ring (2), or a cyclopentane-1,3dione ring (2). This family of compounds has recently been extended with the development of the herbicidal β-triketones derived from a cyclohexane-1,3-dione ring. The cyclohexane-1,3-dione β-triketones, originally synthesized at the Western Research Centre of Zeneca Agrochemical Products, include the herbicide 2-(2-chloro4-mesylbenzoyl)cyclohexane-1,3-dione (sulcotrione) (2) and the tyrosinemia type I therapeutic 2-[2-nitro-4(trifluoromethyl)benzoyl]cyclohexane-1,3-dione (3) (5). An initial observation that few plants grow under the Australian bottlebrush, Calistemon spp, led to the eventual discovery of the synthetic, developmental syncarpic acid herbicides that include 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (1). 1 is an analogue of the naturally occurring syncarpic acids (a syncarpic acid is a 4,4,6,6-tetralkylcyclohexane1,3,5-trione) leptospermone, flavesone, and agglomerone * Author to whom correspondence should be addressed. † Zeneca Central Toxicology Laboratory. ‡ Zeneca Ag Products. § Present address: MRC Toxicology Unit, University of Leicester, PO Box 138, U.K. X Abstract published in Advance ACS Abstracts, December 1, 1995.

0893-228x/96/2709-0024$12.00/0

that have been isolated from steam distillates of Australian plants (2). Evaluation of the 2-benzoylcyclohexane-1,3-dione β-triketones, namely, 2 and 3, in the rat has established that these chemicals interfere with tyrosine catabolism and that 4-hydroxyphenylpyruvate dioxygenase (HPPD)1 is the critical enzyme that is inhibited (6, 7). HPPD catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPPA) to homogentisic acid (HGA), and a consequence of its inhibition in vivo is the appearance of HPPA and its reduced metabolite, (4-hydroxyphenyl)lactate, in urine (7). This is accompanied by a marked increase in plasma tyrosine concentration. An in vitro investigation of the kinetics of inhibition of rat liver HPPD by 2 and 3 has shown these chemicals to be potent, time-dependent, reversible (tight-binding) inhibitors of this enzyme (7) (Figure 1). To extend our knowledge of the mode of action of the β-triketones, we have studied the nature of the inhibition of rat liver HPPD, in vitro, by 1. The present paper documents the finding that this syncarpic acid, like the cyclohexane-1,3-dione β-triketones 2 and 3, is a tightbinding inhibitor of HPPD.

Materials and Methods Chemicals. 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6tetramethylcyclohexane-1,3,5-trione (1), 2-benzoyl-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (4), and 2-(2-carbethoxyacetyl)4,4,6,6-tetramethylcyclohexane-1,3,5-trione (5) with minimum 1 Abbreviations: HPPD, 4-hydroxyphenylpyruvate dioxygenase; HPPA, (4-hydroxyphenyl)pyruvate; HGA, homogentisic acid.

© 1996 American Chemical Society

Inhibition of 4-Hydroxyphenylpyruvate Dioxygenase

Figure 1. Structures of the β-triketones for which the kinetics of inhibition of HPPD have been defined. purities of 95% were supplied as colorless crystalline solids by Zeneca Agrochemical Products, Western Research Centre (Richmond, CA). (4-Hydroxyphenyl)pyruvate, L-amino acid oxidase [L-amino acid:oxygen oxidoreductase (deaminating); EC 1.4.3.2], and ascorbic acid were purchased from Sigma Chemical Co. Ltd. (Poole, Dorset, U.K.). L-[carboxyl-14C]Tyrosine with a specific activity of 54.8 mCi/mmol and a radiochemical purity of 98.7% was purchased from Amersham International plc (Buckinghamshire, U.K.). All other chemicals were of analytical grade or of the highest purity available. Inhibition of 4-Hydroxyphenylpyruvate Dioxygenase (HPPD). Rat liver cytosol (54.6 mg of protein/mL) was used as the source of HPPD (7). Utilization of Oxygen by the Enzyme. This method is a modification of the procedure described by Whelan and Zannoni (8), Coufalik and Monder (9), and Ellis et al. (7). To determine the effect of 1 on HPPD activity, the inhibitor dissolved in aqueous saline (1 dissolved in 0.1 M NaOH and neutralized with HCl) was either preincubated with the enzyme preparation, prior to initiation of the enzyme reaction by the addition of substrate, or coadministered with the substrate. Where the enzyme and inhibitor concentrations were varied, a compensatory change in buffer volume was made to maintain a volume of 4 mL. The Release of Carbon Dioxide from [carboxyl-14C](4Hydroxyphenyl)pyruvate. [carboxyl-14C](4-Hydroxyphenyl)pyruvate was prepared from [carboxyl-14C]tyrosine by the method of Lindstedt and Odelhog (10). HPPD activity was measured using the method of Lindstedt and Odelhog (10) with the exception that rat liver cytosol was used as the source of enzyme and the assay was performed over a 20 min period at 37 °C. Sufficient cytosol was used to ensure that the rate of decarboxylation of substrate was linear under the assay conditions (between 10% and 20% of [carboxyl-14C]HPPA was decarboxylated over a 20 min period at 37 °C). Characterization of the Kinetics of Inhibition of HPPD. The kinetics of inhibition of HPPD was evaluated by measuring changes in dissolved oxygen concentration over a 10 min period following coadministration of varying concentrations of HPPA (400, 200, 60, 50, 40, and 0 µM) and 1 (2, 1, 0.5, 0.25, and 0 µM). These experiments yielded a series of progress curves of oxygen concentration against time (Figure 2). Qualitative investigation of the progress curves suggested the kinetic model shown in Figure 3. This model was translated into a series of differential equations for the evolution in time of active enzyme, inhibitor, oxygen, HPPA, and HGA which were solved numerically. The rate constant of inactivation of HPPD by 1 (k) and other parameters of the model were evaluated by fitting the simulated oxygen profiles to the experimental data (7).

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Figure 2. Progress curves of oxygen utilization with time during the conversion of HPPA to HGA. The reaction mixture contains 0.2 M sodium phosphate buffer (pH 7.2), 1.8 mM ascorbate, rat liver cytosol (2.7 mg of protein/mL of incubate), 0.2 mM HPPA, and 0 (b), 5 ([) 10 (f) or 20 (9) µM 1 in a total volume of 4 mL and were incubated at 37 °C. The enzyme reaction was initiated by the coadministration of HPPA and inhibitor.

Figure 3. Proposed kinetic model for the inhibition of HPPD by 1. E represents the enzyme (HPPD); S, substrate (HPPA); I, inhibitor; ES, the enzyme-substrate complex; P, product (HGA); EI, the enzyme-inhibitor complex; Km, Michaelis constant for HPPD; kp[O2][ES], rate of product formation; k[I][E], the rate of association of the inhibitor with the enzyme; and kd[EI], the rate of dissociation of the inhibitor from the EI complex. Similarly, the kinetics of the inhibition of HPPD was evaluated following coadministration of HPPA (200 and 100 µM) and the syncarpic acids 2-benzoyl-4,4,6,6-tetramethylcyclohexane1,3,5-trione (4) (7.5, 5, 4, 2.5, and 1 mM) and 2-(2-carbethoxyacetyl)-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (5) (200, 150, 100, 50, and 25 µM). Determination of the Dissociation Rate of 1 from the Enzyme-Inhibitor Complex. Studies had established that the catalytic activity of HPPD decreased markedly over several hours at 37 °C, the required time period to measure the rate of dissociation of 1 from the HPPD-inhibitor complex (7). To overcome this, the dissociation of 1 was studied at 25 °C. At this temperature, the enzyme retained >90% of its activity over a sufficient period, 10 h, to measure a recovery of activity. Rat liver cytosol (5.4 mL, 54.6 mg of protein/mL) was incubated with 250 µM 1 (600 µL) at 37 °C for 20 min. This mixture was transferred to a dialysis sac (Medicell International Ltd.) and unbound inhibitor removed against 5 L of 0.2 M TrisHCl buffer (pH 7.4) at 0 °C for 6 h. Immediately following dialysis, oxygen consumption or release of [14C]carbon dioxide from [carboxyl-14C]HPPA by HPPD was negligible, establishing that 1 had not dissociated from the enzyme-inhibitor complex. The enzyme-inhibitor complex was then dialyzed against 0.2 M Tris-HCl buffer (pH 7.4; 600 mL) with stirring at 25 °C, and samples of cytosol (0.5 mL) were taken hourly up to 6 h and stored at -70 °C prior to analysis. Buffer changes (600 mL) were made hourly. The recovery of HPPD activity was determined from three separate experiments by measurement of the release of [14C]carbon dioxide from [carboxyl-14C]HPPA. An identical procedure was conducted (in parallel) with rat liver cytosol that was incubated in the absence of 1 to serve as the control.

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Ellis et al.

Figure 4. Biotransformation of (4-hydroxyphenyl)pyruvic acid (HPPA) to homogentisic acid (HGA) catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPPD).

Figure 6. A representative plot of the effect of enzyme concentration upon the formation of HGA from HPPA. The reaction mixture contains 0.2 M sodium phosphate buffer (pH 7.2), 1.8 mM ascorbate, 0.2 mM HPPA, rat liver cytosol (0.42.3 mg of protein/mL of incubate), and 0 (b), 35 ([), or 50 (9) nM 1 in a total volume of 4 mL and was incubated at 37 °C. Enzyme and inhibitor were incubated together for 3 min prior to the initiation of the enzymatic reaction by the addition of substrate.

Figure 5. Effect of inhibitor concentration on the formation of HGA from HPPA. The reaction mixture contains 0.2 M sodium phosphate buffer (pH 7.2), 1.8 mM ascorbate, 0.2 mM HPPA, rat liver cytosol (2.7 mg of protein/mL of incubate), and 1 (0-200 nM) in a total volume of 4 mL and incubated at 37 °C. Enzyme and inhibitor were incubated together for 3 min prior to the start of the enzymatic reaction by the addition of substrate. In the absence of inhibitor, the rate of oxygen consumption was 0.48 ( 0.03 µL/(mg of protein‚min). Results are expressed as mean ( SD for three consecutive analyses. Under these conditions, the apparent IC50 of compound 1 can be established at approximately 50 nM.

Results and Discussion 4-Hydroxyphenylpyruvate dioxygenase, a key enzyme in the catabolism of tyrosine, is a member of a family of enzymes that catalyze the oxidative decarboxylation of 2-oxoacids with an accompanying hydroxylation. The HPPD catalyzed biotransformation of HPPA to HGA requires an oxidative decarboxylation of the 2-oxopropionate moiety of HPPA to acetate and a hydroxylation of the aromatic ring with 1,2-migration of the acetate group (Figure 4). During the transformation, two oxygen atoms, derived from molecular oxygen, are incorporated into HGA (12). The catalytic activity of HPPD can be assayed by measuring either oxygen utilization by the enzyme or the release of 14CO2 from the labeled substrate [1-14C]HPPA. Rat liver cytosol is used as a convenient source of HPPD. The rate of formation of HGA from HPPA decreased markedly when rat liver cytosol was incubated with 1 for 3 min, prior to the initiation of the enzyme reaction by the addition of substrate. At 200 nM 1, approximately 95% of the enzyme activity was lost and an apparent IC50 was established at approximately 50 nM (Figure 5) (note, caution is required when quoting IC50 values for tightbinding enzyme inhibitors as this value is both time and enzyme concentration dependent). The inhibition of HPPD by 1 is time-dependent; varying the incubation time of 1 (100 nM) with HPPD prior to the addition of substrate resulted in a loss of approximately 80% of enzyme activity at 3 min. At 12 min the capacity for HPPD to catalyze the oxidation of HPPA was almost completely inhibited (the rate of oxygen uptake by HPPD in the absence of 1 was 6.68 ( 0.3 µL of O2/min, data not

Figure 7. Recovery of HPPD activity upon dissociation of 1 from the catalytically inactive enzyme-inhibitor complex. Enzyme activity was established by measuring the release of 14CO2 from [1-14C]HPPA as described by Lindstedt and Odelhog (10). Results are expressed as mean ( SD (n ) 3).

shown). In addition, the effect of HPPD concentration on the initial velocity of the enzyme reaction in the presence of varying amounts of 1 established this chemical to be a potent inhibitor of this enzyme (Figure 6). This was confirmed from progress curve data. A representative progress curve of the change in the rate of oxygen consumption with time for the inhibition of HPPD by 1 is shown in Figure 2. Using this approach, the rate of inactivation of HPPD by 1 (E + I f EI) was calculated at (1.5 ( 0.2) × 10-5 s-1 nM-1. These curves of the effect of 1 on HPPD are indicative of a rapid formation of an EI complex and are consistent with that of an irreversible inhibitor or alternatively that of an inhibitor that dissociates extremely slowly from the EI complex (11). An apparent irreversible inactivation would be detected if the dissociation of the inhibitor from the EI complex was extremely slow (immeasurable over the time course of the assay). To ascertain the kinetics of inhibition, the dissociation of 1 from HPPD (EI f E + I) was assessed at 25 °C over a 6 h period using the oxidative decarboxylation of [carboxyl-14C]HPPA as a measure of enzyme recovery (10). Using this approach, a slow recovery of enzyme activity was measured with time and established that 1 is reversibly bound to HPPD (Figure 7). At 6 h, approximately 5.5 ( 0.6% of the enzyme activity was recovered with an estimated half-life (t1/2, 25 °C) of 101 ( 14 h. These studies establish 1 to be a tight-binding

Inhibition of 4-Hydroxyphenylpyruvate Dioxygenase

inhibitor of HPPD (11). The inhibition is characterized by a rapid, time-dependent inactivation of the enzyme by the formation of a HPPD-inhibitor complex that dissociates extremely slowly with a recovery of enzyme activity. In addition to 1, the kinetics of inhibition of HPPD at 37 °C by the syncarpic acids 2-benzoyl-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (4) and 2-(2-carbethoxyacetyl)-4,4,6,6-tetramethylcyclohexane-1,3,5-trione (5) were studied (Figure 1). These syncarpic acids were competitive inhibitors of HPPD with Ki values of 90 µM and 3.7 µM, respectively. This preliminary data set shows that the substituent at the 2-position of a tetramethylated phloroglucinol ring markedly affects the kinetics of inhibition of HPPD by the syncarpic acid. This substituent effect is exemplified by comparison of the kinetics of inhibition expressed by 1 and 4. With 4, the inhibition is time-independent with the free inhibitor and enzyme in dynamic equilibrium. The inhibition is defined by the equilibrium constant, Ki, that is a measure of the affinity of the inhibitor. With 1, the inhibition is progressive with time (time-dependent); ultimately, complete inhibition is expected even with a very dilute concentration of 1, provided that the dissociation of the inhibitor from the EI complex is extremely slow. The inhibitor effectiveness is here expressed not as an equilibrium constant but as a velocity constant (k) which defines the amount of enzyme inhibited in a given period of time by a certain concentration of inhibitor. For 1, the Ki, if able to be measured, would be extremely large. The difference in the kinetics of inhibition of HPPD by 1 and 4 can be accounted for by a difference in the rate of dissociation of the inhibitor from the EI complex. Thus, substitution about the aromatic ring of 4 markedly affects the binding affinity of the inhibitor to HPPD. Other structural features for the inhibition of HPPD by the triketones have been reported (13). The discovery that the β-triketones 1, 2, and 3 are potent inhibitors of rat liver HPPD in vitro has been extended to the plant enzyme (13). The β-triketones, in particular, 2 and 3, inhibit plant HPPD (13-15). In plants, HPPD is required in the biosynthetic pathway to plastoquinones and tocopherols. A consequence of the inhibition of plant HPPD is the cumulation of tyrosine and to a lesser extent phenylalanine and tryptophan, and a depletion of plastoquinone. This combination of effects, leading to a strong bleaching symptomology and growth retardation, is diagnostic of this new herbicidal mode of action and has been termed the triketone effect (13). The inhibition of rat liver HPPD by the tight-binding β-triketones 2 and 3 has been reported (7, 14). The association rates for these chemicals with HPPD were calculated at (9.9 ( 2.5) × 10-5 s-1 nM-1 and (3.3 ( 0.8) × 10-5 s-1 nM-1, respectively (7). In comparison with the kinetics of inhibition of HPPD by 1, there is an approximately 7-fold difference in the rate of association across this series. The half-lives (t1/2, 25 °C) for the

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dissociation of 1 and 2 from HPPD, measured as a recovery of enzyme activity, were estimated at 63 h (7.5 ( 1.0% of enzyme activity recovered at 6 h) and 10 h (37.5 ( 12% of enzyme activity recovered at 6 h), respectively. This established that the affinity of 1 for HPPD is more akin to that of the tyrosinemia type I therapeutic 3 than that of the herbicide 2. The difference in the dissociation rates of the three β-triketones distinguishes between them and shows 1 and 3 to be more potent inhibitors, having a higher binding affinity to rat liver HPPD, in vitro, than 2.

References (1) Penfold, A. R. (1920) The essential oils of Leptospermum flavescent var. grandiflorum and Leptospermum odoratum. J. Proc. R. Soc. N. S. W. 54, 197-207. (2) Hellyer, R. O. (1968) The occurrence of β-triketones in the steamvolatile oils of some myrtaceous Australian plants. Aust. J. Chem. 21, 2825-2828. (3) Benbakkar, M., Baltas, M., Gorrichon, L., and Gorrichon, J. P. (1989) Synthesis of syncarpic acid and related β-oxo δ-enol lactones via selective O- or C-acylation of preformed enolates. Synth. Commun. 19, 3241-3247. (4) Kalsi, P. S., Singh, J., Crow, W. D., and Chhabra, B. R. (1987) Biomimetic synthesis of 2,3-dioxabicyclo[4,4,0]decanes as plant growth regulators. Phytochemistry 26, 3367-3369. (5) Lindstedt, S. T., Holme, E., Lock, E. A., Hjalmarson, O., and Strandvik, B. (1992) Treatment of hereditary tyrosinaemia type 1 by inhibition of 4-hydroxyphenylpyruvate dioxygenase. Lancet 340, 813-817. (6) Lock, E. A., Ellis, M. K., Provan, W. M., and Smith, L. L. (1994) The effect of NTBC on enzymes involved in tyrosine catabolism in the rat. Toxicologist 14, 826. (7) Ellis, M. K., Whitfield, A. C., Gowans, L. A., Auton, T. R., Provan, W. M., Lock, E. A., and Smith, L. L. (1995) Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)cyclohexane-1,3-dione. Toxicol. Appl. Pharmacol. 133, 12-19. (8) Whelan, D. T., and Zannoni, V. G. (1974) Microassay for tyrosine aminotransferase and p-hydroxyphenylpyruvic acid oxidase in mammalian liver and patients with hereditary tyrosinaemia. Biochem. Med. 9, 19-31. (9) Caufalik, A. H., and Monder, C. (1980) Regulation of tyrosine oxidising system in foetal rat liver. Arch. Biochem. Biophys. 199, 67-75. (10) Lindstedt, S. T., and Odelhog, B. (1987) 4-Hydroxyphenylpyruvate dioxygenase from Pseudomanas. Methods Enzymol. 142, 143147. (11) Morrison, J. F., and Walsh, C. T. (1988) The behaviour and significance of slow-binding enzyme inhibitors. Adv. Enzymol. 61, 201-301. (12) Lindblad, B., Lindstedt, G., and Lindstedt, S. T. (1970) The mechanism of enzymic formation of homogentisate from phydroxyphenylpyruvate. J. Am. Chem. Soc. 92, 7446-7449. (13) Prisbylla, M. P., Onisko, B. C., Shribbs, J. M., Adams, D. O., Liu, Y., Ellis, M. K., Hawkes, T. R., and Mutter, L. C. (1993) The novel mechanism of action of the herbicidal triketones. Brighton Crop Prot. Conf.sWeeds 2, 731-738. (14) Schulz, A., Ort, O., Beyer, P., and Kleinig, H. (1993) SC-0051, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase. FEBS Lett. 318, 162-166. (15) Secor, J. (1994) Inhibition of barnyardgrass 4-hydroxyphenylpyruvate dioxygenase by Sulcotrione. Plant Physiol. 106, 1429-1433.

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