The residues of Site 2 are shown as sticks with dot surfaces

The residues of Site 2 are shown as sticks with dot surfaces. Our modeling also predicts that swapping the ester moiety of 1 1 with a less bulky substituent such as chlorine would eliminate any preference for the Grp94 binding pocket. degradation, which are both markers of Hsp90 inhibition, even at the highest levels tested (75 M). We determined the 2 2.7 ? co-crystal structure of Grp94N:2. In this structure, the conformation GGTI-2418 adopted by Grp94 bound with 2, and the overall pose of 2, is identical to that of Grp94N:1, confirming the modeling predictions that led to the design of 2 (Figure 6A). We also determined the 1.9 GGTI-2418 ? structure of 2 soaked into crystals of apo Hsp90N. The high concentration of the protein in the crystal (23 mM) and the soaked ligand (5 mM) allowed us to visualize very low affinity interactions that are beyond the range of detection of solution binding techniques such as ITC. Nevertheless, the overall quality of the electron density for the ligand was poorer than that observed for other complexes in this study, and correlates with the weaker observed binding of 2 to Hsp90. Compared to the structure of Hsp90N:1, the structure of Hsp90N:2 shows that the resorcinol ring of 2, for which there is strong electron density (Figure S1), is rotated in the ATP binding pocket by ~9 in order to accommodate the bulkier methyl ester group (Figure 6B,D). This rotation of the resorcinol results in an unfavorable positioning of the benzyl imidazole moiety so that it no longer occupies Site 1, but instead makes only weak van der Waals interactions with Asp54, Ala55, and Lys58 of helix 2 (Figure 6B,C). The rotation of the resorcinol ring and the potential inability to access Site 1 may account for the poor binding of 2 to Hsp90. Open in a separate window Figure 6 Binding of ligands 2 and 3 in Grp94 and Hsp90. A) Superposition of Grp94N:1, Grp94N:2, and Grp94N:3 showing similar poses for the ligand. B) The binding pose of 2 in Hsp90 is distorted. Overview alignment of Hsp90:1 (green) and Hsp90:2 (cyan) TNFRSF10D crystal structures showing the good superposition of the two protein structures except for the region between residues 103 and 111. The benzyl imidazole groups from 1 (grey) and 2 (magenta) are found in different poses in the two structures. C) Superposition of Grp94N:2 and Hsp90N:2 showing the different poses for the bound ligand and potential clashes between the Grp94 pose and the Hsp90 pocket. D) The resorcinol scaffolds of 1 1 (gray) and 2 (magenta) make similar hydrogen bonds in the Hsp90 ATP binding pocket but 2 is rotationally displaced by about 9 degrees in order to prevent clashes between the larger methyl ester group and Phe138, which blocks Site 2 in Hsp90. E) Phe199 in Grp94 exposes Site 2 in Grp94N:3 despite having the smaller chloro moieties on the resorcinol ring. The residues of Site 2 are shown as sticks with dot surfaces. Our modeling also predicts that swapping the ester moiety of 1 1 with a less bulky substituent such as chlorine would eliminate any preference for the Grp94 binding pocket. We therefore also tested the bis-chloro (3) derivative of 1 1 and measured the binding to each paralog by ITC. As seen in Figure 5C,E, the less bulky 3 binds to Hsp90 and Grp94 with Kds of 0.32 M and 0.65 M, respectively, indicating that this compound is not selective for either paralog, as predicted. Thus, the steric restriction imposed by the bulkier bis-ester moieties is a key determinant that imparts Grp94 selectivity to the resorcinol scaffold. We determined the co-crystal structure of Grp94N:3. Interestingly, as seen in Figure 6E, despite replacement of the bulky bis-ester moiety with the smaller chloro group, Phe199, the gatekeeper residue for Site 2, is rotated into the position that exposes Site 2. This is the same conformation of Phe199 seen in the Grp94N:1 and Grp94N:2 structures, and suggests that the movement of GGTI-2418 Phe199 that exposes Site 2 is not driven by the size of the resorcinylic substitutents at the 3 position. Discussion and Conclusions In this report, we show that the structures of 1 1 in complex with the N domains of Hsp90 and Grp94 reveal two binding poses for the chlorinated resorcinylic moiety of the inhibitor:.