Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel human coronavirus. Currently, no effective antiviral agents exist against this type of virus. A cell-based assay, with SARS virus and Vero E6 cells, was developed to screen existing drugs, natural products, and synthetic compounds to identify effective anti-SARS agents. Of >10,000 agents tested, ≈50 compounds were found active at 10 μM; among these compounds, two are existing drugs (Reserpine 13 and Aescin 5) and several are in clinical development. These 50 active compounds were tested again, and compounds 2–6, 10, and 13 showed active at 3 μM. The 50% inhibitory concentrations for the inhibition of viral replication (EC ₅₀ ) and host growth (CC ₅₀ ) were then measured and the selectivity index (SI = CC ₅₀ /EC ₅₀ ) was determined. The EC ₅₀ , based on ELISA, and SI for Reserpine, Aescim, and Valinomycin are 3.4 μM (SI = 7.3), 6.0 μM (SI = 2.5), and 0.85 μM (SI = 80), respectively. Additional studies were carried out to further understand the mode of action of some active compounds, including ELISA, Western blot analysis, immunofluorescence and flow cytometry assays, and inhibition against the 3CL protease and viral entry. Of particular interest are the two anti-HIV agents, one as an entry blocker and the other as a 3CL protease inhibitor ( K _i = 0.6 μM).

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis‐type and trans‐type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5‐epi‐aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL^pro) to cleave the virus-encoded polyproteins. We report here that the 3CL^pro containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln^-1-Ser¹ site followed by the C-terminal cleavage at the Gln³⁰⁶-Gly³⁰⁷ site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K_d = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K_d = 17.2 nm) or C-terminal amino acids (K_d = 5.6 nm). The data indicate that immature 3CL^pro can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL^pro from the polyprotein and design of new structure-based inhibitors.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-Å resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S₂ subsite, and do not bind in the caspase primary aspartic acid binding pocket (S₁). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

SARS‐CoV is the causative agent of severe acute respiratory syndrome (SARS). The virally encoded 3C‐like protease (3CLPro) has been presumed critical for the viral replication of SARS‐CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM) and 3‐isotheaflavin‐3‐gallate (TF2B) (IC50 = 7 µM). These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro‐inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—)‐epigallocatechin gallte (EGCg), epicatechin (EC), theophylline (TP), catechin (C), epicatechin gallate (ECg) and epigallocatechin (EGC) did not inhibit 3CLPro activity. Only theaflavin‐3,3′‐digallate (TF3) was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.