Optimization and Validation

The optimal concentrations of Hsp90a and TRAP1 used in the assays were determined by concentration-activity titration experiments (Figure 5.4A). Hsp90a at 1 pM and at 0.5 pM produced Pi

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FIGURE 5.4 Hsp90 ATPase assay. (A) Hsp90a and TRAP1 concentration-activity titration. ATPase assays were set up as described in Section 5.5. Hsp90a was at final concentrations of 1 |M and 0.5 |M, and TRAP1 was at final concentrations of 0.5, 0.25, and 0.125 |M. ATP and MDCC-PBP were at 500 |M and 14 |M, respectively, for both proteins. Plates were read out through 4 hr. (B) Inhibition of Hsp90a and TRAP1 ATPase activity by GA and 17AAG. Compounds were diluted serially into DMSO before adding to the assays. The final DMSO concentration was 2.5%. For all experiments, 0.5 |M Hsp90a and 0.125 |M TRAP1 were used with 500 |M ATP and 14 |M MDCC-PBP. Readings were taken through 4 hr. Percent inhibitions were calculated from the rate (slope) of Pi production in the presence and absence of inhibitors. Background samples contained ATP and MDCC-PBP but no protein.

within the detection limits of the MDCC-PBP through the course of the assay. TRAP1 showed a stronger ATPase activity than Hsp90a. At 0.5 and 0.25 pM, TRAP1 quickly produced Pi at levels beyond the maximum detection limit of the added MDCC-PBP. In order to obtain the most sensitive assay possible, the lowest concentration of protein that produced a two- to threefold signal-to-background ratio was chosen for the finalized protocol. For subsequent assays, Hsp90a was used at final 0.5 pM and TRAP1 at final 0.125 pM concentrations.

Using this assay, we determined the potencies of GA and 17AAG to inhibit the ATPase activity of Hsp90a and TRAP1. As shown in Figure 5.4B, both 17AAG and GA significantly inhibited ATPase of Hsp90a, approaching 80% inhibition at 10 pM. In contrast, 17AAG was much less potent on TRAP1 compared with GA, achieving only 12% inhibition at 10 pM (versus >80% inhibition by GA). This was the first demonstration that the two closely related analogs exerted differential activities against TRAP1. Whether this will translate into differences in cellular activity remains to be investigated.

Under similar conditions, we also readily assessed the ATPase activity of Hsp90^ in the absence or presence of compound treatment (data not shown). We were not, however, able to detect any ATPase activity with purified full-length Grp94 despite the clear demonstration of an ATP binding pocket in the crystal structure (Soldano et al. 2003). The data supported the prevailing notion that Grp94 was capable of binding but not able to hydrolyze ATP.

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