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, Ϸ50 compounds were found active at 10 M; 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 M. The 50% inhibitory concentrations for the inhibition of viral replication (EC50) and host growth (CC50) were then measured and the selectivity index (SI ‫؍‬ CC50͞EC50) was determined. The EC50, based on ELISA, and SI for Reserpine, Aescim, and Valinomycin are 3.4 M (SI ‫؍‬ 7.3), 6.0 M (SI ‫؍‬ 2.5), and 0.85 M (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 (Ki ‫؍‬ 0.6 M).

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide alpha,beta-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 microM), the ketomethylene isosteres and tripeptide alpha,beta-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 microM). The Phe-Phe dipeptide inhibitors 18a-e are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 microM is obtained by condensation of the Phe-Phe dipeptide alpha,beta-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18 microM.

Using d-xylose as an appropriate chiral precursor, we have synthesized active neuraminidase inhibitor oseltamivir, antiflu drug Tamiflu, and novel phosphonate congeners that exhibit even stronger antiflu activities by inhibiting the neuraminidases of the wild-type and H274Y mutant of H1N1 and H5N1 viruses. Molecular modeling of the neuraminidase−phosphonate complex indicates a pertinent binding mode of the phosphonate with three arginine residues in the active site. Discovery of such potent neuraminidase inhibitors will offer an opportunity to the development of new anti-influenza drugs.

Severe acute respiratory syndrome (SARS) is caused by a newly emerged coronavirus that infected more than 8000 individuals and resulted in more than 800 fatalities in 2003. Currently, there is no effective treatment for this epidemic. SARS-3CL(pro) has been shown to be essential for replication and is thus a target for drug discovery. Here, a class of stable benzotriazole esters was reported as mechanism-based inactivators of 3CL(pro), and the most potent inactivator exhibited a k(inact) of 0.0011 s(-1) and a K(i) of 7.5 nM. Mechanistic investigation with kinetic and mass spectrometry analyses indicates that the active site Cys145 is acylated, and that no irreversible inactivation was observed with the use of the C145A mutant. In addition, a noncovalent, competitive inhibition became apparent by using benzotriazole ester surrogates in which the bridged ester-oxygen group is replaced with carbon.

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Human enterovirus (EV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. A peptidomimetic inhibitor AG7088 was developed to inhibit the 3C protease of rhinovirus (a member of the family), a chymotrypsin-like protease required for viral replication, by forming a covalent bond with the active site Cys residue. In this study, we have prepared the recombinant 3C protease from EV71 (TW/2231/98), a particular strain which causes severe outbreaks in Asia, and developed inhibitors against the protease and the viral replication. For inhibitor design, the P3 group of AG7088, which is not interacting with the rhinovirus protease, was replaced with a series of cinnamoyl derivatives directly linked to P2 group through an amide bond to simplify the synthesis. While the replacement caused decreased potency, the activity can be largely improved by substituting the alpha,beta-unsaturated ester with an aldehyde at the P1' position. The best inhibitor 10b showed EC(50) of 18 nM without apparent toxicity (CC(50)>25 microM). Our study provides potent inhibitors of the EV71 3C protease as anti-EV71 agents and facilitates the combinatorial synthesis of derivatives for further improving the inhibitory activity.

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