Objective and Rationale

A homogeneous and sensitive HTRF binding assay was developed to allow prosecution of an HTS campaign for novel small molecule Hsp90 inhibitors. The HTRF assay was based on a non-radioactive resonance energy transfer between a donor label (europium chelate) and an acceptor label (allophycocyanin [APC]) brought into close proximity by a specific binding interaction.

Ex 340 nm

Ex 340 nm

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.

Fluorescent lanthanide (e.g., europium [Eu]) chelates with long excited state lifetimes were used to avoid interference caused by short-lived emission from acceptor molecules that were directly excited rather than excited by energy transfer. In this assay, the APC-streptavidin and Eu-anti-His antibody were brought together by binding of biotin-GA to the His-tagged N terminal ATP domain (amino acids 9 to 236) of Hsp90a (N-Hsp90a-His) as shown in Figure 5.1A. Excitation of Eu chelate at 340 nm caused APC to produce a unique signal at 665 nm. This assay measured the ability of compounds to disrupt the binding interaction between biotin-GA and Hsp90a, which caused the loss of energy transfer from the donor Eu chelate to the acceptor APC.

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