Sivaraman Sharada, Sullivan Todd J, Johnson Francis, Novichenok Polina, Cui Guanglei, Simmerling Carlos, Tonge Peter J
Department of Chemistry, SUNY at Stony Brook, Stony Brook, New York 11794-3400, and Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, New York 11794-8651.
J Med Chem. 2004 Jan 29;47(3):509-18. doi: 10.1021/jm030182i.
To explore the molecular basis for the picomolar affinity of triclosan for FabI, the enoyl reductase enzyme from the type II fatty acid biosynthesis pathway in Escherichia coli, an SAR study has been conducted using a series of triclosan analogues. Triclosan (1) is a slow, tight-binding inhibitor of FabI, interacting specifically with the E.NAD(+) form of the enzyme with a K(1) value of 7 pM. In contrast, 2-phenoxyphenol (2) binds with equal affinity to the E.NAD(+) (K(1) = 0.5 microM) and E.NADH (K(2) = 0.4 microM) forms of the enzyme and lacks the slow-binding step observed for triclosan. Thus, removal of the three triclosan chlorine atoms reduces the affinity of the inhibitor for FabI by 70,000-fold and removes the preference for the E.NAD(+) FabI complex. 5-Chloro-2-phenoxyphenol (3) is a slow, tight-binding inhibitor of FabI and binds to the E.NAD(+) form of the enzyme (K(1) = 1.1 pM) 7-fold more tightly than triclosan. Thus, while the two ring B chlorine atoms are not required for FabI inhibition, replacement of the ring A chlorine increases binding affinity by 450,000-fold. Given this remarkable observation, the SAR study was extended to the 5-fluoro-2-phenoxyphenol (4) and 5-methyl-2-phenoxyphenol (5) analogues to further explore the role of the ring A substituent. While both 4 and 5 are slow, tight-binding inhibitors, they bind substantially less tightly to FabI than triclosan. Compound 4 binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3.2 and 240 nM, respectively, whereas compound 5 binds exclusively to the E.NADH enzyme complex with a K(2) value of 7.2 nM. Thus, the ring A substituent is absolutely required for slow, tight-binding inhibition. In addition, pK(a) measurements coupled with simple electrostatic calculations suggest that the interaction of the ring A substituent with F203 is a major factor in governing the affinity of analogues 3-5 for the FabI complex containing the oxidized form of the cofactor.
为了探究三氯生对大肠杆菌II型脂肪酸生物合成途径中的烯酰还原酶FabI具有皮摩尔亲和力的分子基础，我们使用一系列三氯生类似物进行了构效关系（SAR）研究。三氯生（1）是FabI的一种缓慢、紧密结合的抑制剂，它与该酶的E.NAD(+)形式特异性相互作用，K(1)值为7 pM。相比之下，2-苯氧基苯酚（2）与该酶的E.NAD(+)（K(1)=0.5 microM）和E.NADH（K(2)=0.4 microM）形式具有相同的亲和力，并且没有观察到三氯生所具有的缓慢结合步骤。因此，去除三氯生的三个氯原子会使抑制剂对FabI的亲和力降低70000倍，并消除对E.NAD(+) FabI复合物的偏好。5-氯-2-苯氧基苯酚（3）是FabI的一种缓慢、紧密结合的抑制剂，它与该酶的E.NAD(+)形式结合（K(1)=1.1 pM），比三氯生紧密7倍。因此，虽然B环上的两个氯原子对于抑制FabI不是必需的，但A环氯原子的取代使结合亲和力增加了450000倍。鉴于这一显著发现，构效关系研究扩展到了5-氟-2-苯氧基苯酚（4）和5-甲基-2-苯氧基苯酚（5）类似物，以进一步探究A环取代基的作用。虽然4和5都是缓慢、紧密结合的抑制剂，但它们与FabI的结合比三氯生弱得多。化合物4与该酶的E.NAD(+)和E.NADH形式结合，K(1)和K(2)值分别为3.2和240 nM，而化合物5仅与E.NADH酶复合物结合，K(2)值为7.2 nM。因此，A环取代基对于缓慢、紧密结合抑制是绝对必需的。此外，pK(a)测量结合简单的静电计算表明，A环取代基与F203之间的相互作用是决定类似物3 - 5对含有辅因子氧化形式的FabI复合物亲和力的主要因素。
