INHIBITION OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE BY RO-4-TRIFLUOROMETHYLBENZOYL)-CYCLOHEXANE-1,3-DIONE AND RO-4-METHANESULFONYLBENZOYL)-CYCLOHEXANE-1,3-DIONE

The administration of the compound 2-(2-nitro-4-trifluoromethylbenzoyl )-cyclohexane- 1,3-dione (NTBC) to rats(10 mg/ kg body wt) caused an e levation in the concentration of plasma tyrosine and gave products in urine that were identified as 4-hydroxyphenylpyruvate (HPPA) and 4-hyd roxyphenyllactate (HPLA). This observed chemically induced tyrosinemia established that this compound perturbs tyrosine catabolism and sugge sted that the causal effect is the inhibition of 4-hydroxyphenylpyruva te dioxygenase (HPPD). This was confirmed when rat liver HPPD was foun d to be markedly inhibited by NTBC when the enzyme and chemical were i ncubated, in vitro, for 3 min at 37 degrees C prior to the initiation of the enzyme reaction by the addition of substrate. At 100 nM NTBC, a pproximately 90% of the enzyme activity was lost and an IC50 was calcu lated at approximately 40 nM. The inhibition of HPPD by NTBC (50 nM) i s time-dependent; the enzyme activity was reduced by >50% within 30 se c. Progress curve data of loss of enzyme activity with time gave a rat e constant for the inactivation of rat liver HPPD [k, formation of an HPPD-inhibitor (EI) complex] by NTBC of 9.9 +/- 2.5 x 10(-5) sec(-1) nM(-1). Ii was established that NTBC is not irreversibly bound in the EI complex but slowly dissociates with a recovery of enzyme activity o f 13.7 +/- 1.0% over a 7-hr period (t 1/2, 25 degrees C estimated at 6 3 hours). In comparison, the compound ro-4-methanesulfonylbenzoyl)-cyc lohexane-1,3-dione (CMBC), an analog of NTBC, gave a similar rate for the inactivation of HPPD (k, 3.3 +/- 0.8 x 10(-5) sec(-1) nM(-1)), wh ereas 45 +/- 8% of the enzyme activity was recovered over a 7-hr perio d (t 1/2, 25 degrees C approximately 10 hr). These studies establish t hat NTBC and CMBC are potent, time-dependent (tight-binding) reversibl e inhibitors of HPPD. The inhibition is characterized by a rapid inact ivation of the enzyme by the formation of an HPPD-inhibitor complex th at dissociates with recovery of enzyme activity. In vivo, the inhibiti on of HPPD causes a tyrosinemia that abates with the recovery of enzym e activity. The understanding of the mechanism by which NTBC perturbs tyrosine catabolism has led to the clinical use of this chemical as th e first effective pharmacological therapy for the hereditary disorder tyrosinemia I. (C) 1995 Academic Press, Inc.