Validation and Optimization

For any HTRF binding assay, it is desirable to achieve saturating concentrations with detection reagents. Under those conditions, each binding event would produce a transfer of energy and

GA 16 Radicicol 2 7

GA 16 Radicicol 2 7

FIGURE 5.1 HTRF biochemical binding assay for Hsp90 inhibitors. (a) HTRF assay using biotin-GA and N-Hsp90a-His. (b) Determination of binding potency (IC50) of GA and radicicol for N-Hsp90a-His by HTRF assay set up as described in Section 5.2. N-Hsp90a-His and biotin-GA were at final 100 nM and 30 nM concentrations, respectively.

generation of signal. Ideally, a matrix of all four reagents involved (biotin-GA, APC-streptavidin, Eu-anti-His, and N-Hsp90a-His) would produce the optimal concentrations for each of the components. However, this was not practical, as a matrix of such complexity and magnitude might be unwieldy except to the most highly skilled practitioner. A more workable approach would be to fix the concentration of one or two parameters and vary the others.

To make optimization of the Hsp90 HTRF assay easier and practical, the concentrations of Hsp90 and its binding partner were initially held constant and the detection reagents were varied. Certain concepts were known or had to be assumed. For example, we knew that each streptavidin molecule could bind more than one biotin molecule and the affinity was very strong in the picomolar range. Therefore, it was assumed that adding equal molarities of streptavidin and biotin would result in essentially complete binding of the biotin molecules to streptavidin so that testing a large range of APC-streptavidin combinations was not necessary.

The Eu-anti-His antibody affinity was not provided by the vendor. It was assumed that the antibody affinity was in the lower nanomolar range and was tested in a range starting from low nanomolar to as high as practically could be achieved, taking into consideration the starting concentration of the stock and the overall cost. However, keep in mind that the background signal for an HTRF assay comes from the APC and Eu. Even if saturating levels of detection reagents could be achieved, a balance between the specific binding and the non-specific background signal generation is required.

The reported literature affinity values of GA for Hsp90 were in the 0.1 to 0.4 pM range. As a practical matter, the biotin-GA was initially set at 100 nM with 30 nM Hsp90 in a matrix of APC-SA and Eu-anti-His antibody. This resulted in the best signal-to-background ratio of 40 to 50. The unexpected key to the success of Hsp90 HTRF assays with biotin-GA turned out to be the DTT included as a matter of course in the assay buffer. Removal of the DTT resulted in almost a complete loss of specific signal. We subsequently discovered that DTT actually converted GA to its reduced dihydro-GA form that was recently reported to exhibit substantially higher (~25-fold) binding affinity for Hsp90 (Maroney et al. 2006).

Refinement of the assay consisted of lowering the Hsp90 in matrixes of varying detection reagents to find the lowest protein concentration (for the most sensitive assay) that still produced a good signal-to-background ratio. Ultimately, the optimal concentrations of each component were determined to be 30 nM for Hsp90a, 100 nM for biotin-GA, 5 nM for Eu-anti-His, and 40 nM for APC-streptavidin. Next, we confirmed that at optimal concentrations of biotin-GA, Eu-anti-His, and APC-streptavidin, the assay response was linear to Hsp90 concentration (up to 30 nM). Finally, under optimal conditions, we determined the potencies of the reference compounds: GA and radicicol, another known natural product inhibitor of Hsp90 (Figure 5.1B). It was notable that the observed IC50 value of 16 nM for GA in the presence of DTT already reached the lower detection limit of the HTRF assay (~15 nM). The actual binding affinity of the reduced form of GA for Hsp90 could be higher (i.e., IC50 < 16 nM), as reported by Maroney et al. in 2006 (5 nM, measured by ThermoFluor microcalorim-etry) and determined by a more sensitive AlphaScreen™ assay described in Section 5.4.

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