Cellbased Mechanistic Assays 561 Objectives and Rationale

A number of cell-based assays were developed to evaluate the cellular effect of Hsp90 inhibition. While intracellular Hsp90 ATPase activity cannot be readily measured, several surrogate response markers have been identified based on unique cellular reactions to Hsp90 inhibition. The chaperone cycle of Hsp90-client protein interaction is driven by ATP loading and hydrolysis and requires recruitment of various co-chaperones such as p23 at different stages of the cycle (Kamal, Boehm, and Burrows 2004). The co-chaperone p23 is important for catalytic Hsp90 activity and its association with Hsp90 is mandatory for overall chaperoning activity. The Hsp90-p23 interaction requires ATP binding but not ATP hydrolysis. In addition, p23 specifically recognizes the Hsp90-ATP complexes and not Hsp90 alone. An ATP-competitive small molecule inhibitor prevents binding of ATP to Hsp90 and causes rapid dissociation of p23 from hsp90.

Consequently, client proteins become unstable and eventually undergo proteasome-dependent degradation. On the other hand, an Hsp90 inhibitor also elicits a compensatory stress response via induction of Hsp70. This is accomplished by Hsp90 inhibitor-mediated activation of heat shock factor (HSF) either directly via dissociation of HSF from the inhibitory Hsp90, and/or indirectly via accumulation of destabilized client proteins. Collectively, Hsp90-p23 dissociation, client depletion, and Hsp70 upregulation are considered signature responses indicative of Hsp90 inhibition, and form the molecular basis of target modulation assays for Hsp90 inhibitors in cells.

Using TRF in-cell Western technology, we developed cell-based assays for routine determination of the potencies of compounds to downregulate client proteins (Raf, c-Met, etc.) and induce Hsp70. The traditional Western blot analysis is time consuming, labor intensive, low throughput and at best, semi-quantitative. The in-cell Western (Figure 5.5A) detects target proteins in their cellular environment, and thereby eliminates the need for cell lysate preparation, electrophoresis, transfer

IP: p23

Hsp90

I p23

Downregulation of c-Met

A 17AAG

Downregulation of c-Met

A 17AAG

Induction of Hsp70

A 17AAG

Induction of Hsp70

A 17AAG

[Drug], Log M

FIGURE 5.5 (A) In-cell Western target modulation assay. (B) Potency of 17AAG to downregulate client protein (c-Met) and induce hsp70. GTL-16 gastric tumor cells were treated with various concentrations of 17AAG for 20 hr. IC50 and EC50 values for 17AAG to cause c-Met degradation or Hsp70 induction were determined by in-cell Western analysis. (C) Dissociation of Hsp90-p23 complex by 17AAG. GTL-16 cells were treated with 17AAG (500 nM) for various times. Whole cell lysates were collected after treatment and subjected to immunoprecipitation (IP) of p23. The amount of p23 bound to Hsp90 was determined by Western blot analysis of the immunoprecipitate.

of protein to membrane, and film development. Moreover, the in-cell Western performed in 96-well microplates allows quantitative and accurate determination of cellular potency (IC50 or EC50) of compounds in a medium or high-throughput manner. A large number of compounds can be evaluated in multiple cell lines for effects on the same or different target proteins in a single experiment. For selected inhibitors, we also evaluated their ability to disrupt Hsp90-p23 interaction using IP (immunoprecipitation) Western blot analysis (a medium-throughput ELISA assay is currently in development but will not be discussed here).

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