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

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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.