Bihovsky R, Powers J C, Kam C M, Walton R, Loewi R C
Department of Medicinal Chemistry, Berlex Laboratories, Cedar Knolls, New Jersey 07927.
J Enzyme Inhib. 1993;7(1):15-25. doi: 10.3109/14756369309020184.
Analogs of Ep-475 (2a), designed to explore the role played by the carboxylate in epoxysuccinate thiol protease inhibitors, have been synthesized and tested as inhibitors of papain and cathepsin B. Papain and cathepsin B are rapidly inactivated by carboxylates 2a and 6a, but are inactivated much more slowly by 2b-2f, 6c, and 6f, in which the carboxylate is absent or replaced by an amide, ester, or ketone. This order of reactivity contrasts with the inherent reactivity of substituted epoxides toward a non-enzymatic thiolate, previously shown to decrease in the order: COCH3 > CO2CH3 > CONH2 > H > CO2H. The results suggest that electrostatic attraction between the carboxylate of the inhibitor and protonated His159 of papain facilitates docking of the inhibitor in the active site of the enzyme, a conclusion reached previously from X-ray crystallographic structures of epoxysuccinates bound to papain. The most reactive isoleucine analog, 6a, was significantly less reactive than leucine-containing Ep-475 (2a), while the less reactive isoleucine derivatives, 6c and 6f, were similar in reactivity to the corresponding leucine derivatives, 2c and 2f, respectively.
为了探究环氧琥珀酸酯硫醇蛋白酶抑制剂中羧酸盐所起的作用而设计的Ep - 475（2a）类似物已被合成，并作为木瓜蛋白酶和组织蛋白酶B的抑制剂进行了测试。木瓜蛋白酶和组织蛋白酶B可被羧酸盐2a和6a迅速灭活，但被2b - 2f、6c和6f灭活的速度要慢得多，其中羧酸盐不存在或被酰胺、酯或酮取代。这种反应活性顺序与取代环氧化物对非酶硫醇盐的固有反应活性形成对比，先前已表明其反应活性顺序为：COCH3 > CO2CH3 > CONH2 > H > CO2H。结果表明，抑制剂的羧酸盐与木瓜蛋白酶质子化的His159之间的静电吸引促进了抑制剂在酶活性位点的对接，这一结论先前是从与木瓜蛋白酶结合的环氧琥珀酸酯的X射线晶体结构得出的。反应活性最高的异亮氨酸类似物6a的反应活性明显低于含亮氨酸的Ep - 475（2a），而反应活性较低的异亮氨酸衍生物6c和6f的反应活性分别与相应的亮氨酸衍生物2c和2f相似。
