Optimization and Validation

To obtain meaningful IC50 values, it was important to show that a decrease in protein concentration produces a proportional drop in assay signal. Assays were set up as described above. N-Hsp90a-His

NA NA NA NA NA NA

NA NA NA NA NA NA

C

110

90

n

.2

70

2

X

50

£

o

30

a?

100 nM Biotin-radicicol

50 nM Biotin-radicicol

25 nM Biotin-radicicol

30000 20000 10000

100 nM Biotin-radicicol

30000 20000 10000

30000 20000 10000 0

50 nM Biotin-radicicol

30000 20000 10000 0

15000 10000 5000 0

25 nM Biotin-radicicol

15000 10000 5000 0

10 20 30 [N-Hsp90a] nM

FIGURE 5.2 TRF binding assay for Hsp90 inhibitors. (A) TRF binding assay using biotin-radicicol and N-Hsp90a-His. NA = neutravidin. Eu = europium. (B) Titration of N-Hsp90a-His. TRF assays were set up as described in Section 5.3. N-Hsp90a-His was diluted to 60, 30, and 15 nM and combined 1:1 with 200, 100, or 50 nM biotin-radicicol. (C) Determination of binding potency (IC50) of GA and 17AAG for N-Hsp90a-His by TRF assays. N-Hsp90a-His and biotin-radicicol were at final 30 nM and 100 nM concentrations, respectively. S/B = signal to background.

was diluted to two times the final concentration—60, 30, and 15 nM. The background in this case was no protein rather than 25 pM radicicol. Biotin-radicicol was diluted to 200, 100, and 50 nM and combined at equal volume with the N-Hsp90a to give final biotin-radicicol concentrations of 100, 50, and 25 nM combined with 30, 15, or 7.5 nM N-Hsp90a. Figure 5.2B shows the range of protein concentrations for each concentration of biotin-radicicol that produced a corresponding proportional decrease in signal with each dilution in protein. In all cases this range was from 10 to 30 nM N-Hsp90a. To preserve a high signal-to-background ratio, 30 nM N-Hsp90a and 100 nM biotin-radicicol were chosen for the final assay condition. We then determined the potency of the GA and 17AAG reference compounds (Figure 5.2C). The calculated IC50 values for GA and 17AAG were 69 and 582 nM, respectively—comparable to those reported in the literature in the absence of the DTT reducing agent.

0 0

Post a comment