Ellis M K, Whitfield A C, Gowans L A, Auton T R, Provan W M, Lock E A, Smith L L
Investigative Toxicology Section, Zeneca Central Toxicology Laboratory, Alderley Park, Cheshire, United Kingdom.
Toxicol Appl Pharmacol. 1995 Jul;133(1):12-9. doi: 10.1006/taap.1995.1121.
The administration of the compound 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) to rats (10 mg/kg body wt) caused an elevation in the concentration of plasma tyrosine and gave products in urine that were identified as 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA). This observed chemically induced tyrosinemia established that this compound perturbs tyrosine catabolism and suggested that the causal effect is the inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD). This was confirmed when rat liver HPPD was found to be markedly inhibited by NTBC when the enzyme and chemical were incubated, 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, approximately 90% of the enzyme activity was lost and an IC50 was calculated at approximately 40 nM. The inhibition of HPPD by NTBC (50 nM) is time-dependent; the enzyme activity was reduced by > 50% within 30 sec. Progress curve data of loss of enzyme activity with time gave a rate 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. It was established that NTBC is not irreversibly bound in the EI complex but slowly dissociates with a recovery of enzyme activity of 13.7 +/- 1.0% over a 7-hr period (t1/2, 25 degrees C estimated at 63 hours). In comparison, the compound 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-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), whereas 45 +/- 8% of the enzyme activity was recovered over a 7-hr period (t1/2, 25 degrees C approximately 10 hr). These studies establish that NTBC and CMBC are potent, time-dependent (tight-binding) reversible inhibitors of HPPD. The inhibition is characterized by a rapid inactivation of the enzyme by the formation of an HPPD-inhibitor complex that dissociates with recovery of enzyme activity. In vivo, the inhibition of HPPD causes a tyrosinemia that abates with the recovery of enzyme activity. The understanding of the mechanism by which NTBC perturbs tyrosine catabolism has led to the clinical use of this chemical as the first effective pharmacological therapy for the hereditary disorder tyrosinemia I.
给大鼠（体重10 mg/kg）施用化合物2-(2-硝基-4-三氟甲基苯甲酰基)-环己烷-1,3-二酮（NTBC）后，血浆酪氨酸浓度升高，尿液中出现了被鉴定为4-羟基苯丙酮酸（HPPA）和4-羟基苯乳酸（HPLA）的产物。这种观察到的化学诱导型酪氨酸血症表明该化合物扰乱了酪氨酸分解代谢，并提示其因果效应是对4-羟基苯丙酮酸双加氧酶（HPPD）的抑制。当在体外将大鼠肝脏HPPD与该化学物质于37℃孵育3分钟，然后加入底物启动酶反应时，发现NTBC可显著抑制HPPD，从而证实了这一点。在100 nM NTBC时，约90%的酶活性丧失，计算得出IC50约为40 nM。NTBC（50 nM）对HPPD的抑制作用具有时间依赖性；在30秒内酶活性降低超过50%。酶活性随时间丧失的进程曲线数据得出NTBC使大鼠肝脏HPPD失活（k*，形成HPPD-抑制剂（EI）复合物）的速率常数为9.9±2.5×10⁻⁵秒⁻¹ nM⁻¹。已确定NTBC在EI复合物中并非不可逆结合，而是缓慢解离，在7小时内酶活性恢复13.7±1.0%（25℃时t1/2估计为63小时）。相比之下，NTBC的类似物化合物2-(2-氯-4-甲磺酰基苯甲酰基)-环己烷-1,3-二酮（CMBC）使HPPD失活的速率相似（k*，3.3±0.8×10⁻⁵秒⁻¹ nM⁻¹），而在7小时内45±8%的酶活性得以恢复（25℃时t1/2约为10小时）。这些研究表明NTBC和CMBC是HPPD的强效、时间依赖性（紧密结合）可逆抑制剂。这种抑制的特征是通过形成解离并恢复酶活性的HPPD-抑制剂复合物使酶迅速失活。在体内，HPPD的抑制导致酪氨酸血症，随着酶活性的恢复而减轻。对NTBC扰乱酪氨酸分解代谢机制的理解已使该化学物质在临床上用作遗传性疾病I型酪氨酸血症的首个有效药物治疗。
