Identification of new classes of ricin toxin inhibitors by virtual screening

Abstract

We used two virtual screening programs, ICM and GOLD, to dock nearly 50,000 compounds into each of two conformations of the target protein ricin A chain (RTA). A limited control set suggests that candidates scored highly by two programs may have a higher probability of being ligands than those in a list from a single program. Based on the virtual screens, we purchased 306 compounds that were subjected to a kinetic assay. Six compounds were found to give modest, but significant, inhibition of RTA. They also tended to inhibit Shiga toxin A chain, with roughly the same IC(50). The compounds generally represent novel chemical platforms that do not resemble RTA substrates, as currently known inhibitors do. These six were also tested in a cell-based assay for their ability to protect cells from intact ricin. Two compounds were effective in this regard, showing modest to strong ricin inhibition, but also showing some cytotoxicity. RTA, with its large, polar active site is a difficult drug design target which is expected to bind small molecules only weakly. The ability of the method to find these novel platforms is encouraging and suggests virtual screening can contribute to the search for ricin and Shiga toxin inhibitors.

Figure 1

Solvent-accessible surface structure of RTA. On the surface, oxygens are red, nitrogens blue, and carbons green. A. RTA in the open form with the inhibitor PTA (PDB:1BR6) occupying the adenine specificity pocket, and shown as a stick molecule. B. RTA in the closed form with the inhibitor 2,5-diamino-4,6-dihydroxypyrimidine (PDB:1IL5) shown in stick bonds.

Figure 1

Solvent-accessible surface structure of RTA. On the surface, oxygens are red, nitrogens blue, and carbons green. A. RTA in the open form with the inhibitor PTA (PDB:1BR6) occupying the adenine specificity pocket, and shown as a stick molecule. B. RTA in the closed form with the inhibitor 2,5-diamino-4,6-dihydroxypyrimidine (PDB:1IL5) shown in stick bonds.

Figure 2

Correlation of virtual screening scores against the ChemBridge diversity library. A. Correlation between docking scores for two independent screens using the program ICM; the correlation coefficient is 0.56. B. Correlation scores between programs ICM and GOLD; the correlation coefficient is 0.05(Minetti et al., 2003). The large circles show scores for a positive control of known RTA open form inhibitors.

Figure 2

Correlation of virtual screening scores against the ChemBridge diversity library. A. Correlation between docking scores for two independent screens using the program ICM; the correlation coefficient is 0.56. B. Correlation scores between programs ICM and GOLD; the correlation coefficient is 0.05(Minetti et al., 2003). The large circles show scores for a positive control of known RTA open form inhibitors.

Figure 3

Dose response inhibition of RTA. The data reflect the inhibition of the enzyme by increasing concentrations of CB7543758, dissolved in DMSO.

Figure 4

Dose response inhibition of ricin in a cell based assay. The dashed line shows cell viability after ricin intoxication. Open circles show the dose dependent cytotoxicity of the inhibitor without ricin, and closed circles show the viability of cells challenged with ricin and varying doses of inhibitor.

Figure 4

Dose response inhibition of ricin in a cell based assay. The dashed line shows cell viability after ricin intoxication. Open circles show the dose dependent cytotoxicity of the inhibitor without ricin, and closed circles show the viability of cells challenged with ricin and varying doses of inhibitor.