serotypes tested with a mean EC50 value of 0.822 µM (range: 0.003–8.112 µM) (28). The 50% cytotoxic concentration of AG7088 is > 1,000 µM compared with 150 µM and 77 µM for pirodavir and pleconaril, respectively (28). These studies establish AG7088 as a highly potent, nontoxic antirhinoviral agent with broad efficacy against multiple virus serotypes. The compound has been formulated for intranasal delivery and has recently entered clinical trials.
Experimental Crystal Structure of AG7088 Bound to Serotype 2 Rhinovirus 3C Protease. Serotype 2 human rhinovirus 3C protease was incubated with a 3-fold molar excess of AG7088 in the presence of 2% (vol/vol) DMSO for 24 h at 4°C. The complex was concentrated to 6.8 mg/ml and then passed through a 0.22-µm cellulose-acetate filter. Crystals were grown at 13°C by using a hanging-drop vapor-diffusion method in which equal volumes (3 µl) of the protein-ligand complex and reservoir solution were mixed on plastic coverslips and sealed over individual wells filled with 1 ml of reservoir solution containing 20% (vol/vol) polyethylene glycol (molecular weight 10,000) and 0.1 M Hepes (pH 7.5).
A single crystal measuring 0.3×0.1×0.1 mm (space group P212121; a=34.32, b=65.68, c=77.89 Å) was prepared for low-temperature data collection by transfer to an artificial mother liquor solution consisting of 400 µl of the reservoir solution mixed with 125 µl of glycerol and then flash frozen in a stream of N2 gas at –170°C. X-ray diffraction data were collected with a MAR Research 345-mm imaging plate and processed with DENZO. Diffraction data were 89.2% complete to a resolution of 1.85 Å with R(sym)=1.9%. Protein atomic coordinates from the cocrystal structure of type 2 3C protease with compound I (15) were used to initiate rigid-body refinement in X-PLOR followed by simulated annealing and conjugate gradient minimization protocols. Placement of the inhibitor, addition of ordered solvent, and further refinement proceeded as described in ref. 15. The final R factor was 21.8% [12,184 reflections with F > 2s(F)]. The root-mean-square deviations from ideal bond lengths and angles were 0.016 Å and 2.9°, respectively. The final model consisted of all atoms for residues 1–180 (excluding the side chain of residues 12, 21, 45, and 65) plus 221 water molecules.
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