Liu Yaya, Saldivar Ayda, Bess Joshua, Solomon Larry, Chen Chih-Ming, Tripathi Rakesh, Barrett Leo, Richardson Paul L, Molla Akhteruzzaman, Kohlbrenner William, Kati Warren
Antiviral Department, Infectious Disease Research and Advanced Technology, Pharmaceutical Discovery, Abbott Laboratories, Abbott Park, Illinois 60064-6217, USA.
Biochemistry. 2003 Jul 29;42(29):8862-9. doi: 10.1021/bi034661v.
Indandiones were identified as a novel class of small molecule inhibitors of hepatitis C virus NS3 serine protease from high throughput screening. We further studied the structure activity relationships and the mechanisms of inhibition for this class of compounds. Our studies revealed two similar, yet different, mechanisms accounting for the apparent indandione inhibition of HCV NS3 protease. In one case, the apparent inhibition results from the chemical breakdown of the parent compound and the subsequent redox chemistry of the compound. Oxidation of the cysteine containing substrate A to a disulfide-linked dimer converts this substrate to a potent, slow-binding inhibitor with a K(i) value of 170 nM. The second class of indandiones appears to react directly with the substrate to form an S-phenyl disulfide adduct with the P1 cysteine. This modification converts the substrate to a slow-binding inhibitor with a K(i) value of 110 nM, a k(on) = 2370 M(-1) s(-1), and k(off) = 2.5 x 10(-4) s(-1). A stable analogue of this latter compound was synthesized that contained a CH(2)-S linkage instead of the S-S linkage. The CH(2)-S compound showed no inhibition at concentrations as high as 40 microM, which suggests an important role for the S-S linkage in the inhibitory mechanism. Cysteine 159, which lies near the active site of the HCV protease, was mutated to serine. The C159S mutant displayed wild-type catalytic activity and susceptibility to inhibition by the S-S linked inhibitor. This result argues against a mechanism involving disulfide exchange between the inhibitor and the sulfhydryl group of C159. The mechanism of inhibition for this S-S linked substrate based inhibitor is likely due to oxidation of cysteines involved in chelation of the structural zinc atom.
茚二酮类化合物是通过高通量筛选鉴定出的一类新型丙型肝炎病毒NS3丝氨酸蛋白酶小分子抑制剂。我们进一步研究了这类化合物的构效关系和抑制机制。我们的研究揭示了两种相似但又不同的机制，可解释茚二酮类化合物对丙型肝炎病毒NS3蛋白酶的明显抑制作用。在一种情况下，明显的抑制作用源于母体化合物的化学分解以及该化合物随后的氧化还原反应。含半胱氨酸的底物A氧化为二硫键连接的二聚体，将该底物转化为一种强效的慢结合抑制剂，其抑制常数（Ki）值为170 nM。第二类茚二酮类化合物似乎直接与底物反应，与P1位半胱氨酸形成S-苯基二硫醚加合物。这种修饰将底物转化为一种慢结合抑制剂，其Ki值为110 nM，结合速率常数（kon）= 2370 M-1 s-1，解离速率常数（koff）= 2.5×10-4 s-1。合成了后一种化合物的稳定类似物，其含有CH2-S键而非S-S键。CH2-S化合物在高达40 μM的浓度下未显示出抑制作用，这表明S-S键在抑制机制中起重要作用。位于丙型肝炎病毒蛋白酶活性位点附近的半胱氨酸159突变为丝氨酸。C159S突变体表现出野生型催化活性，且对S-S键连接的抑制剂敏感。该结果排除了抑制剂与C159巯基之间发生二硫键交换的机制。这种基于S-S键连接底物的抑制剂的抑制机制可能是由于参与结构锌原子螯合的半胱氨酸被氧化。
