The Middle-East Respiratory Syndrome coronavirus (MERS-CoV) causes severe acute pneumonia and renal failure. The MERS-CoV papain-like protease (PL(pro)) is a potential target for the development of antiviral drugs. To facilitate these efforts, we determined the three-dimensional structure of the enzyme by X-ray crystallography. The molecule consists of a ubiquitin-like domain and a catalytic core domain. The catalytic domain displays an extended right-hand fold with a zinc ribbon and embraces a solvent-exposed substrate-binding region. The overall structure of the MERS-CoV PL(pro) is similar to that of the corresponding SARS-CoV enzyme, but the architecture of the oxyanion hole and of the S3 as well as the S5 specificity sites differ from the latter. These differences are the likely reason for reduced in vitro peptide hydrolysis and deubiquitinating activities of the MERS-CoV PL(pro), compared to the homologous enzyme from the SARS coronavirus. Introduction of a side-chain capable of oxyanion stabilization through the Leu106Trp mutation greatly enhances the in vitro catalytic activity of the MERS-CoV PL(pro). The unique features observed in the crystal structure of the MERS-CoV PL(pro) should allow the design of antivirals that would not interfere with host ubiquitin-specific proteases.

We have determined the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3C pro ). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3C pro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3C pro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted us to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compounds. We synthesized a series of peptidic ␣,␤-unsaturated ethyl esters of increasing length and for each inhibitor candidate, we determined a crystal structure of its complex with the EV68 3C pro , which served as the basis for the next design round. To exhibit inhibitory activity, compounds must span at least P3 to P1=; the most potent inhibitors comprise P4 to P1=. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concentration [EC 50 ] ‫؍‬ 0.5 M for the best compound). Antiviral activities were determined using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC 50 s of Ϸ180 nM against EV71 and Ϸ60 nM against human rhinovirus 14 in a live virus-cell-based assay. Even the shorter SG75, spanning only P3 to P1=, displayed significant activity (EC 50 ‫؍‬ 2 to 5 M) against various rhinoviruses.

FhuF is a cytoplasmic 2Fe-2S protein of Escherichia coli loosely associated with the cytoplasmic membrane. E. coli fhuF mutants showed reduced growth on plates with ferrioxamine B as the sole iron source, although siderophore uptake was not defective in transport experiments. Removal of iron from coprogen, ferrichrome, and ferrioxamine B was significantly lower in fhuF mutants compared to the corresponding parental strains, which suggested that FhuF is involved in iron removal from these hydroxamate-type siderophores. A redox potential E(1/2) of -310 +/- 25 mV relative to the normal hydrogen electrode was determined for FhuF by EPR redox titration; this redox potential is sufficient to reduce the siderophores coprogen and ferrichrome. Mössbauer spectra revealed that FhuF in its [Fe(2+)-Fe(3+)] state is also capable of direct reduction of ferrioxamine B-bound ferric iron, thus proving its reductase function. This is the first report on a bacterial siderophore-iron reductase which in vivo seems to be specific for a certain group of hydroxamates.

The main proteinase (M(pro)) of the severe acute respiratory syndrome (SARS) coronavirus is a principal target for the design of anticoronaviral compounds. Benzotriazole esters have been reported as potent nonpeptidic inhibitors of the enzyme, but their exact mechanism of action remains unclear. Here we present crystal structures of SARS-CoV M(pro), the active-site cysteine of which has been acylated by benzotriazole esters that act as suicide inhibitors. In one of the structures, the thioester product has been hydrolyzed and benzoic acid is observed to bind to the hydrophobic S2 pocket. This structure also features the enzyme with a shortened N-terminal segment ("amputated N finger"). The results further the understanding of the important role of the N finger for catalysis as well as the design of benzotriazole inhibitors with improved specificity.