Development of SARS virus inhibitor using unusual amino acid-containing natural products as seeds

严重急性呼吸综合征（SARS）冠状病毒的3C样蛋白酶（3CL）是一种巯基蛋白酶，是SARS冠状病毒成熟的关键酶。在从相应的MBP（麦芽糖结合蛋白）-His（六组氨酸）-Flag标签融合蛋白生产成熟3CL蛋白酶的过程中，我们鉴定了两个由成熟3CL蛋白酶降解而得的片段蛋白，并首次发现成熟SARS 3CL蛋白酶在188 Arg/189 Gln位点发生降解，导致催化活性丧失。188位Arg突变为Ile显着提高了蛋白酶的稳定性，产生的R188 I突变蛋白酶可以消化保守的十一肽底物，其效率为K_m = 33.8μM，k_<cat>= 4753 s^<-1>。在突变蛋白酶的C-末端添加His标签，使其催化活性降低到亲本蛋白酶的0.03，然后合成含有不寻常氨基酸的天然产物，寻找具有SARS 3CL蛋白酶抑制剂活性的种子化合物。作为合适的候选化合物，我们选择了从海绵中分离的五肽衍生物miraziridine A及其截短类似物。为了构建miraziridine A的骨架，侧链未保护的乙烯基精氨酸与通过常规固相方法制备的含aziridine的片段缩合。缺少乙烯基精氨酸位点的类似物显示出与咪唑啶A相当的抑制活性，而缺少氮丙啶位点的类似物对组织蛋白酶B（一种典型的巯基蛋白酶）显示出非常弱的抑制活性。（CH_2CH_2CON（CH_3）_2）-CHO对R188 I突变型3CL蛋白酶的催化活性有一定的抑制作用，而E-64对突变型3CL蛋白酶无抑制作用。

The 3C-like (3CL) protease, a thiol protease, of severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the maturation of SARS coronavirus. In the production of mature 3CL protease from the corresponding MBP (maltose binding protein)-His (six histidine)-Flag tag-fused protein, we identified two fragment proteins derived from degradation of the mature 3CL protease, and found for the first time that mature SARS 3CL protease is subject to degradation at the 188Arg/189Gln site, resulting in loss of catalytic activity. Mutation of Arg at the 188 position to Ile remarkably increased the stability of the protease, and the resulting R188I mutant protease could digest the conserved undecapeptide substrate with the efficiency of K_m = 33.8μM and k_<cat> = 4753 s^<-1>. Addition of His tag to the C-terminus of the mutant protease decreased the catalytic activity to 0.03 of the parent protease.We then achieved syntheses of natural products containing unusual amino acids to search seeds compounds those can act as SARS 3CL-protease inhibitor. As a suitable candidate, compound, we selected miraziridine A, a pentapeptide derivative isolated from marine sponge, and its truncated analogs. To construct the backbone of miraziridine A, a side-chain-unprotected vinylogous arginine was condensed with an aziridine-containingfragment prepared by a conventional solid-phase procedure. An analog lacking the vinylogous arginine site showed comparable inhibitoryactivity with miraziridine A, whereas an analog lacking the aziridine site showed remarkably weak inhibitory activity for cathepsin B, a typical thiol protease.We also find that a substrate-based tetra-peptide aldehyde, Ac-Ala-Val-Leu-NHCH (CH_2CH_2CON(CH_3)_2)-CHO, moderately inhibited the catalytic activity of the R188I mutant 3CL protease, whereas E-64, a typical cysteine protease inhibitor, showed no inhibitory activity for the mutant 3CL protease.